Cellular Permissivity Factor for Viruses and Uses Thereof

ABSTRACT

The present invention provides methods and compositions related to the generation of host cells permissive for virus growth, particularly Porcine Reproductive and Respiratory Syndrome (PRRS) virus.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.12/775,283, filed May 6, 2010, an allowed application, which is acontinuation of U.S. application Ser. No. 11/113,751, filed Apr. 25,2005, now U.S. Pat. No. 7,754,464, and claims benefit of U.S.Provisional Patent Application Ser. Nos. 60/565,214, filed Apr. 23,2004, and 60/634,736, filed Dec. 9, 2004. The complete text anddisclosure of the 12/775,283 application is incorporated by referenceherein as if fully set forth.

FIELD OF THE INVENTION

The present invention provides methods and compositions related to thegeneration of host cells permissive for virus growth for viruses of thefamily Asfarviridae and Arteriviridae.

BACKGROUND OF THE INVENTION Asfarviridae

Asfarviridae is a family of icosohedral enveloped viruses whose genomeconsists of a single molecule of linear double-stranded DNA of about150000-190000 nucleotides long. The name of the family is derived fromAfrican Swine Fever and Related Viruses. African Swine Fever Virus(ASFV) is the type species of the Asfivirus genus and is the sole memberof the family. Recently, porcine CD163 polypeptide has been surmised byimplication to be the cellular receptor for African swine fever virus(ASFV) (Sanchez-Tones et al., 2003)

Arteriviridae

Viruses of the family of Arteriviridae includes equine arteritis virus(EAV), lactate dehydrogenase-elevating virus (LDV) and simianhemorrhagic fever virus (SHFV). The Arterivirus having the greatesteconomic importance is Porcine Reproductive and Respiratory SyndromeVirus (PRRSV).

PRRSV

Porcine Reproductive and Respiratory Syndrome (PRRS) is one of the mosteconomically important diseases of swine. The syndrome appeared almostsimultaneously in North America and in Western Europe in the late 1980s,and has since spread to become endemic in the major swine producingnations of Europe, Asia, and the Americas. The etiologic agent of PRRSis a virus that has been designated PRRS virus or PRRSV. For bothEuropean and North American PRRS, the disease is characterized byreproductive failure in sows and gilts (late term abortions, stillbirths, and mummies), high mortality among nursery pigs, and respiratorydisease in swine of all ages. The disease has been the subject of recentreviews (Mengeling and Lager, 2000; Murtaugh et al., 2002; Nodelijk,2002; Plagemann, 2003).

In the pig, PRRSV infection is limited to a subset of cells of themonocyte/macrophage lineage. Fully differentiated porcine alveolarmacrophage (PAM) cells are the primary target cells for viralreplication (Duan et al., 1997a; Duan et al., 1997b). Immortalization ofPAM cells is technically challenging, and when successful, has resultedin cell lines that are not permissive for PRRS virus growth (Weingartlet al., 2002). PRRS virions are specifically bound by macrophages andinternalized in clathrin-coated pits by endocytosis. Release fromendocytic vesicles requires acidic pH (Nauwynck et al., 1999). Initialbinding of virions is mediated by interaction of the viral matrixprotein with heparin sulfate glycosaminoglycans (Delputte et al., 2002).Internalization can be facilitated by a 210 or 220 kDa membraneglycoprotein, as incubation of PAM cells with monoclonal antibodies tothis polypeptide blocks infection with PRRS virus (Duan et al., 1998;Wissink et al., 2003). The 210 kDa glycoprotein has recently beenidentified as sialoadhesin, a member of siglec family of sialic acidbinding immunoglobulin-like lectins (Pensaert et al., 2003).Transfection of the non-permissive PK-15 (porcine kidney) cell line withporcine sialoadhesin conferred the ability to internalize PRRSVparticles, but there remained an apparent block at the uncoating stage,as virions entered into cellular vesicles but did not undergonucleocapsid disintegration and vesicle membrane fusion. Viral geneswere not expressed, and the transfected PK-15 cells were not renderedpermissive for the PRRS virus (Vanderheijden et al., 2003). To ourknowledge, transfection with sialoadhesin has not been shown to besufficient to convert any PRRSV non-permissive cell line to aPRRSV-permissive phenotype.

Apart from primary porcine cells of the monocyte/macrophage lineage, theonly other cell type known to be permissive for the growth of PRRSV incell culture is the immortalized monkey kidney cell line MA-104(Chladeket al., 1998) and derivatives such as MARC-145 (Kiln et al., 1993) andCL-2621. It is not known why this one particular cell line ispermissive, yet other mammalian cell lines are not. The PRRS virus bindsspecifically to a number of different cell types, but does not initiateinfection (Kreutz, 1998; Therrien et al., 2000). In MARC-145 cells, theinternalization of the virus by endocytosis and subsequent uncoating inlow pH vesicles seems to mimic similar events in PAM cells (Kreutz andAckermann, 1996). However, a number of monoclonal antibodies that bindto porcine sialoadhesin fail to detect a homologous protein on thesurface of MARC-145 cells (Duan et al., 1998; Wissink et al., 2003),suggesting that MARC-145 cells may use a divergent member of the sameprotein family or a different receptor altogether.

Current PRRSV vaccines are propagated on simian cell lines, which is apotentially dangerous activity. The use of simian cell lines for vaccineproduction has the potential to introduce primate viruses ofsignificance into swine lines intended for xenotransplant purposes.Because swine are being increasingly explored as a source ofxenotransplanted organs for humans, the introduction of primate celllines to swine populations may ultimately pose a risk to humansreceiving xenotransplanted organs. Thus, it would be prudent to avoidthe use of simian cell lines in swine vaccine preparations. It would betherefore desirable to identify or generate non-simian cells or celllines capable of supporting PRRSV replication. Towards this goal, it isessential to identify the gene product(s) which may be responsible forconferring the permissivity for PRRSV replication as seen in certainsimian cells lines as well as PAM cells. Once such gene products areidentified, non-permissive cells might be rendered permissive by, forexample, transfection of the essential gene into them, thereby affordinga wider array of production lines for a vaccine.

One lab has reported that the tetraspanin protein CD151 from MARC-145cells, when transfected into non-permissive BHK-21 cells, conferspermissivity to the PRRS virus (Kapil and Shanmukhappa, 2003;Shanmukhappa and Kapil, 2001). This observation has yet to be confirmedby an independent lab.

We describe here an unrelated polypeptide, which when introduced intonon-permissive cells, confers permissivity to the PRRS virus.

REFERENCES CITED

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SUMMARY OF THE INVENTION

The invention includes a method of facilitating infection of one or morecells by a virus that is selected from the group consisting ofArteriviridae and Asfarviridae, which comprises the step of increasingexpression of a CD163 polypeptide within said cell. In a preferredembodiment the CD163 is membrane bound. In one embodiment the virus isselected from the group consisting of Arteriviridae. In a preferredembodiment the vines is PRRSV. In another embodiment said virus isequine arteritis virus (EAV). In yet another embodiment said virus isAfrican Swine Fever virus (ASFV).

Increased expression of a CD163 polypeptide may be accomplished bymethods such as introduction of exogenous nucleic acids encoding CD163polypeptides. Such methods include but are not limited to transfection,electroporation and fusion with a carrier of a polynucleotide comprisinga polynucleotide encoding a CD163 polypeptide. Increased expression mayalso be accomplished by induction of expression of endogenous CD163 bychemical treatment.

The method may render previously non PRRSV-permissive cells to be PRRSVpermissive. The method may render a cell that was previously PRRSVpermissive to be more PRRSV permissive. The method also includesrendering one or more cells that previously did not express a CD163polypeptide into cells that are induced to express a CD 163 polypeptide.The method also includes rendering one or more cells that previouslyexpressed a CD163 polypeptide to express a higher level of CD 163polypeptide.

The cells in a preferred embodiment are animal cells. They may bevertebrate or invertebrate cells. The cells may be mammalian. The cellsor cell line may be an insect cell line. The cells may be BHK21 cells.The cells may be derived from porcine kidney cells. The cells or cellline may be derived from feline kidney cells. The cells or cell line maybe but are not limited to BHK-21, NLST-1, NLFK-1, Vero, swine testicle,or rabbit lung cells. The cells or cell line may be derived from aviancells. The PRRSV may be of the European or North American genotype.

As noted above, increased expression of a CD163 polypeptide may beaccomplished by methods which include but are not limited to:transfection, electroporation and fusion with a carrier of apolynucleotide comprising a polynucleotide encoding a CD163 polypeptide.In addition, viral vectors may be used to introduce a polynucleotideencoding a CD163 polypeptide. Any CD163 polypeptides are contemplated.Those containing a transmembrane region are preferred. Exemplarypolynucleotide encoding CD163 polypeptides that may be used in themethod are selected from the group consisting of the polynucleotideslisted below.

One such polynucleotide comprises a polynucleotide encoding apolypeptide having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 2 orfragments thereof.

One such polynucleotide comprises a polynucleotide encoding apolypeptide differing from SEQ ID NO: 2 by no more than 20 conservativeamino acid substitutions.

One such polynucleotide comprises a polynucleotide encoding apolypeptide differing from SEQ ID NO: 2 by no more than 10 conservativeamino acid substitutions.

One such polynucleotide comprises a polynucleotide encoding apolypeptide comprising SEQ ID NO: 2.

One such polynucleotide comprises a polynucleotide with the sequence setforth in SEQ ID NO: 1.

One such polynucleotide comprises a polynucleotide encoding apolypeptide that has at least 70% identity to a polypeptide set forth inSEQ ID NO: 14 or fragments thereof.

One such polynucleotide comprises a polynucleotide encoding apolypeptide that has at least 90% identity to a polypeptide set forth inSEQ ID NO: 14 or fragments thereof.

One such polynucleotide comprises a polynucleotide encoding apolypeptide that has at least 99% identity to a polypeptide set forth inSEQ ID NO: 14 or fragments thereof.

One such polynucleotide comprises a polynucleotide encoding apolypeptide differing from SEQ ID NO: 14 by no more than 15 conservativeamino acid substitutions.

One such polynucleotide comprises a polynucleotide encoding apolypeptide differing from SEQ ID NO: 14 by no more than 10 conservativeamino acid substitutions.

One such polynucleotide comprises a polynucleotide encoding apolypeptide comprising SEQ ID NO: 14.

One such polynucleotide comprises a polynucleotide with the sequence setforth in SEQ ID NO: 13.

One such polynucleotide comprises a polynucleotide encoding apolypeptide that has at least 90% identity to a polypeptide set forth inSEQ ID NO: 19 or fragments thereof.

One such polynucleotide comprises a polynucleotide encoding apolypeptide differing from SEQ ID NO: 19 by no more than 2 conservativeamino acid substitutions.

One such polynucleotide comprises a polynucleotide encoding apolypeptide comprising SEQ ID NO: 19.

One such polynucleotide comprises a polynucleotide with the sequence setforth in SEQ ID NO: 18.

One such polynucleotide comprises a polynucleotide encoding apolypeptide differing from SEQ ID NO: 24 by no more than 2 conservativeamino acid substitutions.

One such polynucleotide comprises a polynucleotide encoding apolypeptide comprising SEQ ID NO: 24.

One such polynucleotide comprises a polynucleotide with the sequence setforth in SEQ ID NO: 23.

One such polynucleotide comprises a polynucleotide encoding apolypeptide having at least 96%, 97%, 98%, or 99% identity to apolypeptide set forth in SEQ ID NO: 27 or fragments thereof.

One such polynucleotide comprises a polynucleotide encoding apolypeptide differing from SEQ ID NO: 27 by no more than 20 conservativeamino acid substitutions.

One such polynucleotide comprises a polynucleotide encoding apolypeptide differing from SEQ ID NO: 27 by no more than 10 conservativeamino acid substitutions.

One such polynucleotide comprises a polynucleotide encoding apolypeptide comprising SEQ ID NO: 27.

One such polynucleotide comprises a polynucleotide with the sequence setforth in SEQ ID NO: 26.

One such polynucleotide comprises a polynucleotide encoding apolypeptide that has at least 98% or 99% identity to a polypeptide setforth in SEQ ID NO: 32 or fragments thereof.

One such polynucleotide comprises a polynucleotide encoding apolypeptide differing from SEQ ID NO: 32 by no more than 15 conservativeamino acid substitutions.

One such polynucleotide comprises a polynucleotide encoding apolypeptide differing from SEQ ID NO: 32 by no more than 10 conservativeamino acid substitutions.

One such polynucleotide comprises a polynucleotide encoding apolypeptide comprising SEQ ID NO: 32.

One such polynucleotide comprises a polynucleotide with the sequence setforth in SEQ ID NO: 31.

One such polynucleotide comprises a polynucleotide encoding apolypeptide that has at least 95%, 96%, 97%, 98%, or 99% identity to apolypeptide set forth in SEQ ID NO: 34 or fragments thereof.

One such polynucleotide comprises a polynucleotide encoding apolypeptide differing from SEQ ID NO: 34 by no more than 15 conservativeamino acid substitutions.

One such polynucleotide comprises a polynucleotide encoding apolypeptide differing from SEQ ID NO: 34 by no more than 10 conservativeamino acid substitutions.

One such polynucleotide comprises a polynucleotide encoding apolypeptide comprising SEQ ID NO: 34.

One such polynucleotide comprises a polynucleotide with the sequence setforth in SEQ ID NO: 33.

One such polynucleotide comprises a polynucleotide encoding apolypeptide that has at least 95%, 96%, 97%, 98%, or 99% identity to apolypeptide set forth in SEQ ID NO: 36 or fragments thereof.

One such polynucleotide comprises a polynucleotide encoding apolypeptide differing from SEQ ID NO: 36 by no more than 15 conservativeamino acid substitutions.

One such polynucleotide comprises a polynucleotide encoding apolypeptide differing from SEQ ID NO: 36 by no more than 10 conservativeamino acid substitutions.

One such polynucleotide comprises a polynucleotide encoding apolypeptide comprising SEQ ID NO: 36.

One such polynucleotide comprises a polynucleotide with the sequence setforth in SEQ ID NO: 35.

One such polynucleotide comprises a polynucleotide encoding apolypeptide that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identity to a polypeptide set forth in SEQ ID NO: 42 orfragments thereof.

One such polynucleotide comprises a polynucleotide encoding apolypeptide differing from SEQ ID NO: 42 by no more than 15 conservativeamino acid substitutions.

One such polynucleotide comprises a polynucleotide encoding apolypeptide differing from SEQ ID NO: 42 by no more than 10 conservativeamino acid substitutions.

One such polynucleotide comprises a polynucleotide encoding apolypeptide comprising SEQ ID NO: 42.

One such polynucleotide comprises a polynucleotide with the sequence setforth in SEQ ID NO: 41.

One such polynucleotide comprises a polynucleotide encoding apolypeptide that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identity to a polypeptide set forth in SEQ ID NO: 44 orfragments thereof.

One such polynucleotide comprises a polynucleotide encoding apolypeptide differing from SEQ ID NO: 44 by no more than 15 conservativeamino acid substitutions.

One such polynucleotide comprises a polynucleotide encoding apolypeptide differing from SEQ ID NO: 44 by no more than 10 conservativeamino acid substitutions.

One such polynucleotide comprises a polynucleotide encoding apolypeptide comprising SEQ ID NO: 44.

One such polynucleotide comprises a polynucleotide with the sequence setforth in SEQ ID NO: 43.

One such polynucleotide comprises a polynucleotide encoding apolypeptide that has at least 95%, 96%, 97%, 98%, or 99% identity to apolypeptide set forth in SEQ ID NO: 46 or fragments thereof.

One such polynucleotide comprises a polynucleotide encoding apolypeptide differing from SEQ ID NO: 46 by no more than 15 conservativeamino acid substitutions.

One such polynucleotide comprises a polynucleotide encoding apolypeptide differing from SEQ ID NO: 46 by no more than 10 conservativeamino acid substitutions.

One such polynucleotide comprises a polynucleotide encoding apolypeptide comprising SEQ ID NO: 46.

One such polynucleotide comprises a polynucleotide with the sequence setforth in SEQ ID NO: 45.

One such polynucleotide comprises a polynucleotide encoding apolypeptide that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identity to a polypeptide set forth in SEQ ID NO: 48 orfragments thereof.

One such polynucleotide comprises a polynucleotide encoding apolypeptide differing from SEQ ID NO: 48 by no more than 15 conservativeamino acid substitutions.

One such polynucleotide comprises a polynucleotide encoding apolypeptide differing from SEQ ID NO: 48 by no more than 10 conservativeamino acid substitutions.

One such polynucleotide comprises a polynucleotide encoding apolypeptide comprising SEQ ID NO: 48.

One such polynucleotide comprises a polynucleotide with the sequence setforth in SEQ ID NO: 47.

The method of facilitating infection described above may furthercomprise the step of producing a culture of virus. The invention alsocomprises the step of isolating the virus from said culture.

The invention further comprises the culture isolated by the methoddescribed above.

The invention further comprises the virus isolated by the methoddescribed above.

Any of the methods described above may further comprise the step ofproducing a PRRSV or other viral vaccine. The vaccine may comprisekilled or live attenuated virus. The invention further comprises thevaccines produced by said method.

The invention also comprises a cell or cell line wherein the ability ofone or more cells to be infected by a virus selected from the groupconsisting of Arteriviridae and Asfarviridae has been modified byincreasing expression of a CD163 polypeptide within said cells. Inanother embodiment the vines is PRRSV. In another embodiment said virusis equine arteritis virus (EAV). In yet another embodiment said virus isAfrican Swine Fever virus (ASFV).

In a preferred embodiment the CD163 polypeptide comprises atransmembrane region. In one embodiment the virus is selected from thegroup consisting of Arteriviridae. In a preferred embodiment the virusis PRRSV. In another embodiment said virus is equine arteritis virus(EAV). In yet another embodiment said virus is African Swine Fever virus(ASFV).

The cell or cell line of the invention may have been previously PRRSVnon-permissive and is rendered PRRSV permissive by directing increasedexpression of a CD163 polypeptide within said cell or cell line. Thecell or cell line of the invention may have been previously PRRSVpermissive and is rendered to be more PRRSV permissive by directingincreased expression of a CD163 polypeptide within said cell or cellline.

The cell or cell line of the invention includes cells or cell lines thatdid not express a CD163 polypeptide and is induced to express a CD163polypeptide. The cell or cell line of the invention includes cells orcell lines that previously expressed a CD163 polypeptide and is inducedto express a higher level of CD163 polypeptide.

The cells in a preferred embodiment are animal cells. They may bevertebrate or invertebrate cells. The cells may be mammalian. The cellor cell line may be an insect cell or cell line. The cells may be BHK21cells. The cells may be derived from porcine kidney cells. The cell orcell line may be derived from feline kidney cells. The cells may be, butare not limited to, BHK-21, NLST-1, NLFK-1, Vero, swine testicle (ST),or rabbit lung (RL) cells. The PRRSV may be North American or European.

The invention includes a method for measuring the propensity of a testcell or cell line to allow infection by a virus selected from the groupconsisting of Arteriviridae and Asfarviridae comprising:

a) providing a sample containing nucleic acids from the test cell orcell line;b) determining the amount of polynucleotide encoding a CD163 polypeptideor its complement in said sample;wherein an increased amount of polynucleotide encoding a CD163polypeptide relative to a control sample derived from a control cell orcell line known not to support the growth of said virus indicates apropensity of the test cell or cell line to support the replication ofsaid virus.

In one embodiment the virus is selected from the group consisting ofArteriviridae. In a preferred embodiment the virus is PRRSV. In anotherembodiment said virus is equine arteritis virus (EAV). In yet anotherembodiment said virus is African Swine Fever virus (ASFV).

The amount of polynucleotide encoding a CD163 polypeptide may bedetermined by hybridization.

The amount of polynucleotide encoding a CD163 polypeptide may bedetermined by PCR.

The invention also includes a method for measuring the propensity of atest cell or cell line to allow infection by a virus selected from thegroup consisting of Arteriviridae and Asfarviridae comprising:

(a) providing a sample containing polypeptides from the test cell orcell line;(b) determining the amount of CD163 polypeptide in said sample;wherein an increased amount of a CD163 polypeptide relative to a controlsample derived from a control cell or cell line known not to support thegrowth of said virus indicates a propensity of the test cell or cellline to support the replication of said virus.

In one embodiment the virus is selected from the group consisting ofArteriviridae. In a preferred embodiment the virus is PRRSV. In anotherembodiment said virus is equine arteritis virus (EAV). In yet anotherembodiment said virus is African Swine Fever virus (ASFV).

In one embodiment the deterermining is accomplished by contacting aCD163 polypeptide with an antibody specific for the CD163 polypeptide,under conditions wherein the antibody binds the CD163 polypeptide.

The invention includes a method for measuring the propensity of a firstpig to become infected by a virus selected from the group consisting ofArteriviridae and Asfarviridae comprising:

a) providing a sample containing nucleic acids from the pig to betested;b) determining the amount of polynucleotide encoding a CD163 polypeptideor its complement in said sample;wherein an increased amount of polynucleotide encoding a CD163polypeptide relative to the amount of polynucleotide encoding a CD163polypeptide in a sample from a second pig indicates a greater propensityof the first pig to be infected by said virus.

In one embodiment the virus is selected from the group consisting ofArteriviridae. In a preferred embodiment the virus is PRRSV. In anotherembodiment said virus is equine arteritis virus (EAV). In yet anotherembodiment said virus is African Swine Fever virus (ASFV).

In one embodiment the determining is accomplished by hybridization. Inanother embodiment the determining is accomplished by PCR.

The invention also includes a method for measuring the propensity of afirst pig to become infected by a virus selected from the groupconsisting of Arteriviridae and Asfarviridae comprising:

(a) providing a sample containing polypeptides from pig to be tested;(b) determining the amount of CD163 polypeptide in said sample;wherein an increased amount of a CD163 polypeptide relative to theamount of CD163 polypeptide in a sample from a second pig indicates agreater propensity of the first pig to be infected by said virus.

In one embodiment the virus is selected from the group consisting ofArteriviridae. In a preferred embodiment the virus is PRRSV. In anotherembodiment said virus is equine arteritis virus (EAV). In yet anotherembodiment said virus is African Swine Fever virus (ASFV).

In one embodiment the determining is accomplished by contacting a CD163polypeptide with an antibody specific for the CD163 polypeptide, underconditions wherein the antibody binds the CD163 polypeptide.

The invention also includes an isolated polypeptide wherein thepolypeptide is selected from the group consisting of the polypeptidesdescribed below.

Therefore the invention also includes an isolated polypeptide having atleast 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identity with SEQ ID NO: 2 or fragments thereof.

One such polypeptide is a polypeptide differing from SEQ ID NO: 2 by nomore than 20 conservative amino acid substitutions.

One such polypeptide is a polypeptide differing from SEQ ID NO: 2 by nomore than 10 conservative amino acid substitutions.

One such polypeptide comprises SEQ ID NO: 2.

The invention also includes an isolated polypeptide having at least 70%,71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identity to a polypeptide set forth in SEQ ID NO: 14 or fragmentsthereof.

One such polypeptide is a polypeptide differing from SEQ ID NO: 14 by nomore than 15 conservative amino acid substitutions.

One such polypeptide is a polypeptide differing from SEQ ID NO: 14 by nomore than 10 conservative amino acid substitutions.

One such polypeptide comprises SEQ ID NO: 14.

The invention also includes an isolated polypeptide having at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a polypeptideset forth in SEQ ID NO: 19 or fragments thereof.

The invention also includes an isolated polypeptide differing from SEQID NO: 19 by no more than 2 conservative amino acid substitutions.

One such polypeptide comprises SEQ ID NO: 19.

The invention also includes an isolated polypeptide having at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a polypeptideset forth in SEQ ID NO: 24 or fragments thereof.

The invention also includes an isolated polypeptide differing from SEQID NO: 24 by no more than 2 conservative amino acid substitutions.

One such polypeptide comprises SEQ ID NO: 24.

The invention also includes an isolated polypeptide having at least 96%,97%, 98%, or 99% identity to a polypeptide set forth in SEQ ID NO: 27.

One such polypeptide is a polypeptide differing from SEQ ID NO: 27 by nomore than 20 conservative amino acid substitutions. One such polypeptideis a polypeptide differing from SEQ ID NO: 27 by no more than 10conservative amino acid substitutions.

One such polypeptide is a polypeptide comprising SEQ ID NO: 27.

The invention also includes an isolated polypeptide having at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to a polypeptideset forth in SEQ ID NO: 32 or fragments thereof.

One such polypeptide is a polypeptide differing from SEQ ID NO: 32 by nomore than 15 conservative amino acid substitutions.

One such polypeptide is a polypeptide differing from SEQ ID NO: 32 by nomore than 10 conservative amino acid substitutions.

One such polypeptide is a polypeptide comprising SEQ ID NO: 32.

The invention also includes an isolated polypeptide having at least 95%,96%, 97%, 98%, or 99% identity to a polypeptide set forth in SEQ ID NO:34 or fragments thereof.

One such polypeptide is a polypeptide differing from SEQ ID NO: 34 by nomore than 15 conservative amino acid substitutions.

One such polypeptide is a polypeptide differing from SEQ ID NO: 34 by nomore than 10 conservative amino acid substitutions.

One such polypeptide is a polypeptide comprising SEQ ID NO: 34.

The invention also includes an isolated polypeptide having at least 95%,96%, 97%, 98%, or 99% identity to a polypeptide set forth in SEQ ID NO:36 or fragments thereof.

One such polypeptide is a polypeptide differing from SEQ ID NO: 36 by nomore than 15 conservative amino acid substitutions.

One such polypeptide is a polypeptide differing from SEQ ID NO: 36 by nomore than 10 conservative amino acid substitutions.

One such polypeptide is a polypeptide comprising SEQ ID NO: 36.

The invention also includes an isolated polypeptide having at least 95%,96%, 97%, 98%, or 99% identity to a polypeptide set forth in SEQ ID NO:38 or fragments thereof.

One such polypeptide is a polypeptide differing from SEQ ID NO: 38 by nomore than 15 conservative amino acid substitutions.

One such polypeptide is a polypeptide differing from SEQ ID NO: 38 by nomore than 10 conservative amino acid substitutions.

One such polypeptide is a polypeptide comprising SEQ ID NO: 38.

The invention also includes an isolated polypeptide having at least 95%,96%, 97%, 98%, or 99% identity to a polypeptide set forth in SEQ ID NO:40 or fragments thereof.

One such polypeptide is a polypeptide differing from SEQ ID NO: 40 by nomore than 15 conservative amino acid substitutions.

One such polypeptide is a polypeptide differing from SEQ ID NO: 40 by nomore than 10 conservative amino acid substitutions.

One such polypeptide is a polypeptide comprising SEQ ID NO: 40.

The invention also includes an isolated polypeptide having at 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a polypeptide setforth in SEQ ID NO: 42 or fragments thereof.

One such polypeptide is a polypeptide differing from SEQ ID NO: 42 by nomore than 15 conservative amino acid substitutions.

One such polypeptide is a polypeptide differing from SEQ ID NO: 42 by nomore than 10 conservative amino acid substitutions.

One such polypeptide is a polypeptide comprising SEQ ID NO: 42.

The invention also includes an isolated polypeptide having at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a polypeptideset forth in SEQ ID NO: 44 or fragments thereof.

One such polypeptide is a polypeptide differing from SEQ ID NO: 44 by nomore than 15 conservative amino acid substitutions.

One such polypeptide is a polypeptide differing from SEQ ID NO: 44 by nomore than 10 conservative amino acid substitutions.

One such polypeptide is a polypeptide comprising SEQ ID NO: 44.

The invention also includes an isolated polypeptide having at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a polypeptideset forth in SEQ ID NO: 46 or fragments thereof.

One such polypeptide is a polypeptide differing from SEQ ID NO: 46 by nomore than 15 conservative amino acid substitutions.

One such polypeptide is a polypeptide differing from SEQ ID NO: 46 by nomore than 10 conservative amino acid substitutions.

One such polypeptide is a polypeptide comprising SEQ ID NO: 46.Therefore the invention also includes an isolated polypeptide having atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to apolypeptide set forth in SEQ ID NO: 48 or fragments thereof.

One such polypeptide is a polypeptide differing from SEQ ID NO: 48 by nomore than 15 conservative amino acid substitutions.

One such polypeptide is a polypeptide differing from SEQ ID NO: 48 by nomore than 10 conservative amino acid substitutions.

One such polypeptide is a polypeptide comprising SEQ ID NO: 48.

The invention also includes an isolated CD163 polynucleotide whereinsaid polynucleotide is selected from the group consisting of thepolynucleotides enumerated below.

Therefore the invention also includes an isolated polynucleotidecomprising:

(a) a polynucleotide sequence set forth in SEQ ID NOs: 1 or 5;(b) a polynucleotide that encodes a polypeptide that has at least 70%,71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identity and/or similarity to a polypeptide set forth in SEQ ID NO:2 or fragments thereof;(c) a polynucleotide encoding a polypeptide of SEQ ID NO: 2;(d) a polynucleotide which is the complement of any of (a), (b), or (c).

Therefore the invention also includes an isolated polynucleotidecomprising:

(a) a polynucleotide sequence set forth in SEQ ID NOs: 12 or 13;(b) a polynucleotide that encodes a polypeptide that has at least 99%identity and/or similarity to a polypeptide set forth in SEQ ID NO: 14or fragments thereof;(c) a polynucleotide encoding a polypeptide of SEQ ID NO: 14;(d) a polynucleotide that is the complement of any of (a), (b), or (c).

Therefore the invention also includes an isolated polynucleotidecomprising:

(a) a polynucleotide sequence set forth in SEQ ID NOs: 17 or 18;(b) a polynucleotide encoding a polypeptide of SEQ ID NO: 19 orfragments thereof;(c) a polynucleotide which is the complement of (a) or (b).

Therefore the invention also includes an isolated polynucleotidecomprising:

(a) a polynucleotide sequence set forth in SEQ ID NOs: 22 or 23;(b) a polynucleotide that encodes a polypeptide that has at least 99%,99.9% identity to a polypeptide set forth in SEQ ID NO: 24 or fragmentsthereof;(c) a polynucleotide encoding a polypeptide of SEQ ID NO: 24;(d) a polynucleotide which is the complement of any of (a), (b), or (c).

Therefore the invention also includes an isolated polynucleotidecomprising:

(a) a polynucleotide sequence set forth in SEQ ID NOs: 25 or 26;(b) a polynucleotide that encodes a polypeptide that has at least 96%,97%, 98%, or 99% identity and/or similarity to a polypeptide set forthin SEQ ID NO: 27 or fragments thereof;(c) a polynucleotide encoding a polypeptide of SEQ ID NO: 27;(d) a polynucleotide which is the complement of any of (a), (b), or (c).

Therefore the invention also includes an isolated polynucleotidecomprising:

(a) polynucleotide sequence set forth in SEQ ID NOs: 30 or 31;(b) a polynucleotide that encodes a polypeptide that has at least 96%,96.2%, 97%, 98% or 99% identity and/or similarity to a polypeptide setforth in SEQ ID NO: 32 or fragments thereof;(c) a polynucleotide encoding a polypeptide of SEQ ID NO: 32;(d) a polynucleotide which is the complement of any of (a), (b), or (c).

Therefore the invention also includes an isolated polynucleotidecomprising:

(a) polynucleotide sequence set forth in SEQ ID NO: 33;(b) a polynucleotide that encodes a polypeptide that has at least 95%,96%, 97%, 98%, or 99% identity and/or similarity to a polypeptide setforth in SEQ ID NO: 34 or fragments thereof;(c) a polynucleotide encoding a polypeptide of SEQ ID NO: 34;(d) a polynucleotide which is the complement of any of (a), (b), or (c).

Therefore the invention also includes an isolated polynucleotidecomprising:

(a) polynucleotide sequence set forth in SEQ ID NO: 35;(b) a polynucleotide that encodes a polypeptide that has at least 95%,96%, 97%, 98%, or 99% identity and/or similarity to a polypeptide setforth in SEQ ID NO: 36 or fragments thereof;(c) a polynucleotide encoding a polypeptide of SEQ ID NO: 36;(d) a polynucleotide which is the complement of any of (a), (b), or (c).

Therefore the invention also includes an isolated polynucleotidecomprising:

(a) polynucleotide sequence set forth in SEQ ID NO: 37;(b) a polynucleotide that encodes a polypeptide that has at least 95%,96%, 97%, 98%, or 99% identity and/or similarity to a polypeptide setforth in SEQ ID NO: 38 or fragments thereof;(c) a polynucleotide encoding a polypeptide of SEQ ID NO: 38;(d) a polynucleotide that is the complement of any of (a), (b), or (c).

Therefore the invention also includes an isolated polynucleotidecomprising:

(a) polynucleotide sequence set forth in SEQ ID NO: 39;(b) a polynucleotide that encodes a polypeptide that has at least 95%,96%, 97%, 98%, or 99% identity and/or similarity to a polypeptide setforth in SEQ ID NO: 40 or fragments thereof;(c) a polynucleotide encoding a polypeptide of SEQ ID NO: 40;(d) a polynucleotide that is the complement of any of (a), (b), or (c).

Therefore the invention also includes an isolated polynucleotidecomprising:

(a) polynucleotide sequence set forth in SEQ ID NO: 41;(b) a polynucleotide that encodes a polypeptide that has at least 95%,96%, 97%, 98%, or 99% identity and/or similarity to a polypeptide setforth in SEQ ID NO: 42 or fragments thereof;(c) a polynucleotide encoding a polypeptide of SEQ ID NO: 42;(d) a polynucleotide that is the complement of any of (a), (b), or (c).

Therefore the invention also includes an isolated polynucleotidecomprising:

(a) polynucleotide sequence set forth in SEQ ID NO: 43;(b) a polynucleotide that encodes a polypeptide that has at least 95%,96%, 97%, 98%, or 99% identity and/or similarity to a polypeptide setforth in SEQ ID NO: 44 or fragments thereof;(c) a polynucleotide encoding a polypeptide of SEQ ID NO: 44;(d) a polynucleotide that is the complement of any of (a), (b), or (c).

Therefore the invention also includes an isolated polynucleotidecomprising:

(a) polynucleotide sequence set forth in SEQ ID NO: 45;(b) a polynucleotide that encodes a polypeptide that has at least 85%,85.5%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%; 96%, 97%, 98%;or 99% identity and/or similarity to a polypeptide set forth in SEQ IDNO: 46 or fragments thereof;(c) a polynucleotide encoding a polypeptide of SEQ ID NO: 46;(d) a polynucleotide which is the complement of any of (a), (b), or (c).

The invention also includes an isolated polynucleotide comprising:

(a) polynucleotide sequence set forth in SEQ ID NO: 47;(b) a polynucleotide that encodes a polypeptide that has at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity and/orsimilarity to a polypeptide set forth in SEQ ID NO: 48 or fragmentsthereof;(c) a polynucleotide encoding a polypeptide of SEQ ID NO: 49;(d) a polynucleotide which is the complement of any of (a), (b), or (c).

Therefore the invention also includes a CD163 polypeptide in which thetransmembrane region is deleted.

Therefore the invention also includes a polynucleotide encoding a CD163polypeptide in which the transmembrane region is deleted.

In addition to the foregoing, the invention includes, as an additionalaspect, all embodiments of the invention narrower in scope in any waythan the variations specifically mentioned above.

Brief Description of the Sequence Listings

SEQ ID NO: 1—cDNA sequence encoding porcine susCD163v1SEQ ID NO: 2—predicted amino acid sequence of porcine susCD163v1SEQ ID NO: 3—cDNA sequence, Genbank accession number AJ311716SEQ ID NO: 4—predicted amino acid sequence derived from Genbankaccession number AJ311716SEQ ID NO: 5—cDNA sequence of susCD163v1 containing flanking(non-coding) sequenceSEQ ID NO: 6-11—primer sequencesSEQ ID NO: 12—cDNA sequence encoding porcine susCD163v2 containingflanking (non-coding) sequenceSEQ ID NO: 13—cDNA sequence encoding porcine susCD163v2SEQ ID NO: 14—predicted amino acid sequence of porcine susCD163v2SEQ ID NO: 15-16—primer sequencesSEQ ID NO: 17—cDNA sequence encoding human CD163v2 containing flanking(non-coding) sequenceSEQ ID NO: 18—cDNA sequence encoding human CD163v2SEQ ID NO: 19—predicted amino acid sequence of human CD163v2SEQ ID NO: 20-21—primer sequencesSEQ ID NO: 22—cDNA sequence encoding murine CD163v2 containing flanking(non-coding) sequenceSEQ ID NO: 23—cDNA sequence encoding murine CD163v2SEQ ID NO: 24—predicted amino acid sequence of murine CD163v2SEQ ID NO: 25—cDNA sequence encoding murine CD163v3 containing flanking(non-coding) sequenceSEQ ID NO: 26—cDNA sequence encoding murine CD163v3SEQ ID NO: 27—predicted amino acid sequence of murine CD163v3SEQ ID NO: 28-29—primer sequencesSEQ ID NO: 30—cDNA sequence encoding MARC-145 CD163v2 containingflanking (non-coding) sequenceSEQ ID NO: 31—cDNA sequence encoding MARC-145 CD163v2SEQ ID NO: 32—predicted amino acid sequence of MARC-145 CD163v2SEQ ID NO: 33—cDNA sequence encoding Vero cell CD163v2 transcriptSEQ ID NO: 34—predicted amino acid sequence of Vero cell CD163v2SEQ ID NO: 35—cDNA sequence encoding Vero cell CD163v3 transcriptSEQ ID NO: 36—predicted amino acid sequence of Vero cell CD163v3SEQ ID NO: 37—cDNA sequence encoding Vero cell CD163v4 transcriptSEQ ID NO: 38—predicted amino acid sequence of Vero cell CD163v4SEQ ID NO: 39—cDNA sequence encoding Vero cell CD163v5 transcriptSEQ ID NO: 40—predicted amino acid sequence of Vero cell CD163v5SEQ ID NO: 41—cDNA sequence encoding Vero cell CD163v6 transcriptSEQ ID NO: 42—predicted amino acid sequence of Vero cell CD163v6SEQ ID NO: 43—cDNA sequence encoding Vero cell CD163v7 transcriptSEQ ID NO: 44—predicted amino acid sequence of Vero cell CD163v7SEQ ID NO: 45—cDNA sequence encoding canine CD163v2 transcriptSEQ ID NO: 46—predicted amino acid sequence of canine CD163v2SEQ ID NO: 47—cDNA sequence encoding canine CD163v3 transcriptSEQ ID NO: 48—predicted amino acid sequence of canine CD163v3

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Schematic comparison of susCD163v1 with AJ311716

FIG. 2 Amino acid sequence alignment of susCD163v1 (SEQ ID NO: 2) withAJ311716 (SEQ ID NO: 4)

FIG. 3 Nucleotide sequence alignment of susCD163v1 with AJ311716

FIG. 4 Generation of DNA fragments and ligation to place CD163 directlybehind the RSV promoter. Plasmids were digested with either DraIII orDrdI, followed by a blunting reaction with Klenow enzyme. After cleanup, the plasmids were digested with NotI. Gel purification yielded DNAfragments that were subsequently ligated utilizing the cohesive NotItermini. Promoters from RSV (pRSV) and SV40 (pSV40) are indicated witharrows.

FIG. 5 Map of pcDNA3.1 Directional V5/His/TOPO cloning vector

FIG. 6 Three BHK/CMV/v1 cell lines, #3, #5, and #12 and a non-permissiveBHK cell line were infected with PRRSV isolate P129 and stained withSDOW17-FITC. Panel A shows a non-permissive BHK21 cell clone. Panel Bshows BHK/CMV/v1 clone #3. Panel C shows BHK/CMV/v1 clone #5. Panel Dshows BHKICMV/v1 clone #12.

FIG. 7 Three BHK/RSV/v1 cell lines, #2, #3, and #4 were infected withPRRSV isolate P129 and stained with SDOW17-FITC. Panel A showsBHK/RSV/v1 clone #2. Panel B shows BHK/RSV/v1 clone #3. Panel C showsBHK/RSV/v1 clone #4.

FIG. 8 Feline kidney cell lines stably expressing porcine CD163v1,showing PRRSV plaques. Cell lines NLFK-CMV-susCD163v1-G4F andNLFK-CMV-susCD163v1-G4L, both at passage 4, were infected with the P129isolate of North American PRRSV and incubated for 6 days. Monolayerswere fixed with 80% acetone and stained with monoclonal antibodySDOW17-FITC. Phase contrast microscopy (right) shows localized regionsof viral CPE (plaques), while FA detection (left) shows co-localizedviral nucleocapsid antigen.

FIG. 9 Four FK/RSV/v1 cell lines, #1, #2, #3, and #4 were infected withPRRSV isolate P129 and stained with monoclonal antibody SDOW17-FITC.Panel A shows FK/RSVv1 #1 cell clone. Panel B shows FK/RSV/v1 clone #2.Panel C shows FK/RSV/v1 clone #3. Panel D shows FK/RSV/v1 #4.

FIG. 10 PK-CMV-susCD163v1-A10 cells at passage 19, infected with PRRSVisolate P129. Left: The monolayer was fixed with 80% acetone and stainedwith FITC-conjugated monoclonal antibody SDOW17 (Rural TechnologiesInc), which is specific for PRRSV nucleocapsid. Right: The same wellunder bright field illumination, showing cell distribution.

FIG. 11 BHK-CMVScript-susCD163v2-A9 at passage 17 infected with PRRSVisolate P129. The monolayer was fixed with 80% acetone and stained withFITC-conjugated monoclonal antibody SDOW17 (Rural Technologies Inc),which is specific for PRRSV nucleocapsid.

FIG. 12 Three representative examples of the BHK/RSV/v2 cell lines. Thecells were infected with PRRSV isolate P129 and subsequently stainedwith SDOW17-FITC. Panel A shows cell line BHK/RSV/v2 #1, panel B showscell line BHK/RSV/v2 #34, and panel C shows cell line BHK/RSV/v2 #47.

FIG. 13 FK-cDNA3.1D-humCD163v2-A6 at passage 15 infected with PRRSVisolate P129. The monolayer was then fixed with 80% acetone and stainedwith FITC-conjugated monoclonal antibody SDOW17 (Rural TechnologiesInc), which is specific for PRRSV nucleocapsid.

FIG. 14 The amount of progeny PRRSV produced by four recombinant celllines stably expressing susCD163v1, and by MARC-145 cells, wasdetermined in a growth curve experiment using the NVSL 94-3 isolate ofPRRSV. Samples harvested at 12-hour intervals were titrated on MARC-145cell monolayers.

FIG. 15 Flow cytometry analysis of PRRSV infection in the presence ofCD163 specific antibody. BHK-21 cells expressing MARC-145 CD163 fromtransient transfection were incubated with either CD163 specificantibody or normal goat IgG (NGS) and infected with a GFP-expressingPRRSV. Each data point represents the results of triplicate wells.

FIG. 16 Flow cytometry analysis of PRRSV infection in the presence ofCD163 specific antibody. NLFK cells stably expressing human CD163 wereincubated with either CD163 specific antibody or normal goat IgG (NGS)and infected with a GFP expressing PRRSV. At 24 hours post infection thepercentage of GFP expressing infected cells was determined. Each datapoint represents the result from a single well of cells

FIG. 17. Graphical depiction of six alternative splicing variants ofCD163 mRNA recovered from Vero cells. The six variants differ in thepresence or absence of three exons, designated E6, E105, and E83. ExonsE6 and E105 have lengths that are multiples of three, and therefore donot result in a change in reading frame when absent. In contrast, theabsence of E83 results in a shifted reading frame and an alternativeamino acid sequence at the carboxy terminus of the protein (indicated bya hatched pattern in the figure). The hydrophobic transmembrane (TM)region is encoded within E105.

FIG. 18. PK-RSVScript-susCD163v2 #9 cells infected with PRRSV isolateP129. Undiluted supernatant from PRRSV isolate P201 infected PAMs wasused to infect PK-RSVScript-susCD163v2 #9 cells. After two days ofincubation the cells were fixed and stained with monoclonal antibodySDOW17 as described in Example 11.

FIG. 19. FK-RSVScript-susCD163v2 #51 cells infected with PRRSV isolateP129. Undiluted supernatant from PRRSV isolate P201 infected PAMs wasused to infect FK-RSVScript-susCD163v2 #51 cells. Two days postinfection the cells were acetone fixed and stained with monoclonalantibody SDOW17 as described in Example 11.

FIG. 20. Infection of PK-RSVScript-susCD163v2 clone #9 cells with PRRSVisolate P201. Panel A shows a monolayer of cells infected with PRRSVP201 at passage 1, twenty-four hours post infection. Panel B shows amonolayer of cells 2 days post infection with cell free supernatantPRRSV P201 at passage 10.

FIG. 21. NLFK parent cells and one subclone of FK-cDNA3.1D-humCD163v2-A6were examined for the CD163 expression. Cells were fixed in 80% acetoneand reacted with Goat anti-human CD163 (R&D System at 1:200) for onehour following by washing with PBS. For visualization, donkey anti-GoatIgG conjugated with FITC (Biodesign Inc at 1:100) were used. No specificfluorescence was detected in the NLFK parent cells as shown in FIG. 21A.The majority of the FK.A6.A2 subclone showed good fluorescent stainingindicating the presence of CD163 (FIG. 21B).

DETAILED DESCRIPTION OF THE INVENTION General Definitions

Cells and cell lines can be either “virus permissive” or “virusnon-permissive”. For example, a cell or cell line that is viruspermissive is capable of allowing virus infection, subsequentreplication, and virus production. A cell or cell line that is virusnon-permissive is incapable of allowing virus infection, subsequentreplication, and virus production. A cell line that is already somewhatpermissive may be rendered more permissive by the methods of theinvention.

Arteriviridae refers to a family of enveloped, positive-stranded RNAviruses belonging to the order Nidovirales. The family includes lactatedehydrogenase-elevating virus (LDV) of mice, equine arteritis virus(EAV), simian hemorrhagic fever virus (SHFV), and PRRSV.

Asfarviridae is a family of icosohedral, enveloped viruses whose genomesconsist of single molecules of linear double-stranded DNA about150000-190000 nucleotides long. The name of the family is derived fromAfrican Swine Fever And Releted Viruses. African Swine Fever Virus(ASFV) is the type species of the Asfivirus genus and is the sole memberof the family.

The term “PRRSV” or PRRS virus refers to both European and NorthAmerican PRRS virus genotypes. Within each genotype, isolates typicallyshare 85% or higher nucleotide identity. Between genotypes, however, thelevel of sequence identity is only about 60%. The North Americangenotype of PRRSV is exemplified by the prototype isolate VR2332. Thegenomic sequence of VR2332 is found in Genbank accession number U87392,which is incorporated herein by reference. The European genotype ofPRRSV is exemplified by the prototype isolate “Lelystad virus”. Thegenomic sequence of Lelystad virus is found in Genbank accession numberM96262, which is incorporated herein by reference.

The PRRS virus is a member of the family Arteriviridae. The genome ofthe arteriviruses is single-stranded RNA of positive polarity between 12and 16 kb in length, capped at the 5′ end and polyadenylated at the 3′end. Over two-thirds of the genome is dedicated to open reading frames(ORFs) 1a and 1b, which encode the non-structural functions of thevirus. ORF1b is an extension of ORF1a, and is the result of a ribosomalframeshift. ORFs 1a and 1b are translated directly from the genomic RNA.These large polypeptide products are cleaved by viral proteases to yield12 or 13 discrete smaller peptides. The remaining ORFs, which encodeviral structural proteins, are expressed from a series of 3′ co-terminalsubgenomic RNAs (sgRNAs). The sgRNAs are produced by discontinuoustranscription of negative-stranded RNA, such that a common 5′ leadersequence becomes fused to each transcript. The major structural proteinsare the nucleocapsid (N, encoded by ORF7), the matrix protein (M,encoded by ORF6), and the major envelope glycoprotein (GP5, encoded byORF5).

The remaining proteins, GP4 (ORF4), GP3 (ORF3), GP2 (ORF2a), and E(ORF2b) are minor structural components of the virion, whose functionshave not yet been elucidated. The molecular biology of PRRSV has beenthe subject of recent review articles (Dea et al., 2000; Meulenberg,2000; Snijder and Meulenberg, 2001). As used herein, the term “CD163polypeptide” means a protein encoded by a mammalian CD163 gene,including allelic variants containing conservative or non-conservativechanges. A cDNA sequence that encodes a porcine CD163 polypeptide hasbeen reported (Genbank accession number AJ311716). A murine CD163polypeptide has also been reported (Genbank access number AF274883), aswell as multiple human variants, exemplified by Genbank access numbersAAH51281 and CAA80543. We report herein polynucleotides that encodeporcine, human, murine, canine, and african green monkey CD163polypeptides and which comprise the sequences set forth in SEQ ID NO: 1,5, 12, 13, 17, 18, 22, 23, 25, 26, 30, 31, 33, 35, 37, 39, 41, 43, 45,and 47. A “CD163 polypeptide” is a member of the scavenger receptorcysteine-rich (SRCR) family of transmembrane glycoproteins, and isthought to be expressed exclusively on monocytes and macrophages. Oneidentified role of CD163 is to inhibit oxidative tissue damage followinghemolysis by consuming hemoglobin:haptoglobin complexes by endocytosis.The subsequent release of interleukin-10 and synthesis of hemeoxygenase-1 results in antiinflammatory and cytoprotective effects(Philippidis et al., 2004; Graversen et al., 2002). The human CD163 genespans 35 kb on chromosome 12, and consists of 17 exons and 16 introns. Anumber of isoforms of the CD163 polypeptide, including membrane bound,cytoplasmic and secreted types, are known to be generated by alternativesplicing (Ritter et al., 1999). Isoforms that comprise a transmembranedomain are particularly preferred.

A transmembrane domain is characterized by a polypeptide segment of alarger sequence that is exposed on both sides of a membrane. Thecytoplasmic and extracellular domains are separated by at least onemembrane-spanning segment that traverses the hydrophobic environment ofthe lipid bilayer. The membrane-spanning segment is composed of aminoacid residues with nonpolar side chains, usually in the form of an alphahelix. Segments that contain about 20-30 hydrophobic residues are longenough to span a membrane as an alpha helix, and they can often beidentified by means of a hydropathy plot. The predicted transmembranedomain of SEQ ID NOs:2 and 14 are indicated by bolding in thespecification. To determine whether other CD163 sequences contain asimilar sequence feature is easily determined by inspection of thesequence or hydropathy plots. SEQ ID NOs: 37-40 are representative ofvariant CD163 proteins which do not contain a transmembrane domain andtheir encoding nucleic acids.

As used hereinafter, “polynucleotide” generally refers to anypolyribonucleotide or polydeoxyribonucleotide, which may be unmodifiedRNA or DNA or modified RNA or DNA. “Polynucleotides” include, withoutlimitation, single- and double-stranded DNA, DNA that is a mixture ofsingle- and double-stranded regions, single- and double-stranded RNA,RNA that is mixture of single- and double-stranded regions, and hybridmolecules comprising DNA and RNA that may be single-stranded or, moretypically, double-stranded or a mixture of single- and double-strandedregions. In addition, “polynucleotide” refers to triple-stranded regionscomprising RNA or DNA or both RNA and DNA. The term “polynucleotide”also includes DNAs or RNAs containing one or more modified bases andDNAs or RNAs with backbones modified for stability or for other reasons.“Modified” bases include, for example, tritylated bases and unusualbases such as inosine. A variety of modifications may be made to DNA andRNA; thus, “polynucleotide” embraces chemically, enzymatically ormetabolically modified forms of polynucleotides as typically found innature, as well as the chemical forms of DNA and RNA characteristic ofviruses and cells. “Polynucleotide” also embraces relatively shortpolynucleotides, often referred to as oligonucleotides. Polynucleotideand nucleic acid are used synonymously herein.

As used hereinafter, “polypeptide” refers to any peptide or proteincomprising amino acids joined to each other by peptide bonds or modifiedpeptide bonds. “Polypeptide” refers to both short chains, commonlyreferred to as peptides, oligopeptides or oligomers, and to longerchains, generally referred to as proteins. Polypeptides may containamino acids other than the 20 gene-encoded amino acids. “Polypeptides”include amino acid sequences modified either by natural processes, suchas post-translational processing, or by chemical modification techniqueswhich are well known in the art. Such modifications are well describedin basic texts and in more detailed monographs, as well as in avoluminous research literature. Modifications may occur anywhere in apolypeptide, including the peptide backbone, the amino acid side-chainsand the amino or carboxyl termini. It will be appreciated that the sametype of modification may be present to the same or varying degrees atseveral sites in a given polypeptide. Also, a given polypeptide maycontain many types of modifications. Polypeptides may be branched as aresult of ubiquitination, and they may be cyclic, with or withoutbranching. Cyclic, branched and branched cyclic polypeptides may resultfrom post-translation natural processes or may be made by syntheticmethods. Modifications or modified forms include acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cystine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,proteolytic processing, phosphorylation prenylation, racemization,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins such as arginylation, and ubiquitination (see, for instance,Proteins-Structure and Molecular Properties, 2nd Ed., T. E. Creighton,W.H. Freeman and Company, New York, 1993; Wold, F., Post-translationalProtein Modifications: Perspectives and Prospects, pgs. 1-12 inPostranslational Covalent Modification of Proteins, B. C. Johnson, Ed.,Academic Press, New York, 1983; Seifter et al., “Analysis for proteinmodifications and nonprotein cofactors”, Meth Enzymol (1990) 182:626-646and Rattan et al., “Protein Synthesis: Post-translational Modificationsand Aging”, Ann NY Acad Sci (1992) 663:4842).

A “fragment” of a polypeptide as used herein means a segment of at leastabout 25 consecutive amino acids of a polypeptide of the invention. Inanother embodiment, a “fragment” as used herein means a segment of atleast about 50 consecutive amino acids of a polypeptide of theinvention. In another embodiment, a “fragment” as used herein means asegment of at least about 100 consecutive amino acids of a polypeptideof the invention.

As used hereinafter, “isolated” means altered by the hand of man fromthe natural state. If an “isolated” composition or substance occurs innature, it has been changed or removed from its original environment, orboth. For example, a polynucleotide or a polypeptide naturally presentin a living animal is not “isolated,” but the same polynucleotide orpolypeptide separated from the coexisting materials of its natural stateis “isolated”, as the term is employed herein. Therefore “isolated” asused herein and as understood in the art, whether referring to“isolated” polynucleotides or polypeptides, is taken to mean separatedfrom the original cellular environment in which the polypeptide ornucleic acid is normally found. As used herein therefore, by way ofexample only, a transgenic animal or a recombinant cell line constructedwith a polynucleotide of the invention makes use of the “isolated”nucleic acid. Specifically excluded from the definition of isolatedpolynucleotides of the invention are entire isolated chromosomes fromnative host cells from which the polynucleotide was originally derived.

In the disclosure to follow we will often make use of the term“identity” or similarity as applied to the amino acid sequences ofpolypeptides. Percent amino acid sequence “identity” with respect topolypeptides is defined herein as the percentage of amino acid residuesin the candidate sequence that are identical with the residues in thetarget sequences after aligning both sequences and introducing gaps, ifnecessary, to achieve the maximum percent sequence identity, and notconsidering any conservative substitutions as part of the sequenceidentity. Percent sequence identity is determined by conventionalmethods. For example, BLASTP 2.2.6 [Tatusova TA and TL Madden, “BLAST 2sequences—a new tool for comparing protein and nucleotide sequences.”(1999) FEMS Microbiol Lett. 174:247-250.]

Briefly, as noted above, two amino acid sequences are aligned tooptimize the alignment scores using a gap opening penalty of 10, a gapextension penalty of 0.1, and the “blosum62” scoring matrix of Henikoffand Henikoff (Proc. Nat. Acad. Sci. USA 89:10915-10919. 1992).

The percent identity is then calculated as:

$\frac{\left( {{Total}\mspace{14mu} {number}\mspace{14mu} {of}\mspace{14mu} {identical}\mspace{14mu} {matches}} \right)}{\begin{bmatrix}{{{length}\mspace{14mu} {of}\mspace{14mu} {the}\mspace{14mu} {longer}\mspace{14mu} {sequence}} +} \\{{{number}\mspace{14mu} {of}\mspace{14mu} {gaps}\mspace{14mu} {introduced}\mspace{14mu} {into}\mspace{14mu} {the}\mspace{14mu} {longer}}\mspace{25mu}} \\{{sequence}\mspace{14mu} {to}\mspace{14mu} {align}\mspace{14mu} {the}\mspace{14mu} {two}\mspace{14mu} {sequences}}\end{bmatrix}} \times 100$

Percent sequence “similarity” (often referred to as “homology”) withrespect to a polypeptide of the invention is defined herein as thepercentage of amino acid residues in the candidate sequence that areidentical with the residues in the target sequences after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity (as described above), and also considering anyconservative substitutions as part of the sequence identity.

$\frac{\begin{matrix}{{Total}\mspace{14mu} {number}\mspace{14mu} {of}\mspace{14mu} {identical}\mspace{14mu} {matches}\mspace{14mu} {and}} \\{{conservative}\mspace{14mu} {substitutions}}\end{matrix}}{\begin{bmatrix}{{{length}\mspace{14mu} {of}\mspace{14mu} {the}\mspace{14mu} {longer}\mspace{14mu} {sequence}} +} \\{{{number}\mspace{14mu} {of}\mspace{14mu} {gaps}\mspace{14mu} {introduced}\mspace{14mu} {into}\mspace{14mu} {the}\mspace{14mu} {longer}}\mspace{25mu}} \\{{sequence}\mspace{14mu} {to}\mspace{14mu} {align}\mspace{14mu} {the}\mspace{14mu} {two}\mspace{14mu} {sequences}}\end{bmatrix}} \times 100$

Amino acids can be classified according to physical properties andcontribution to secondary and tertiary protein structure. A conservativesubstitution is recognized in the art as a substitution of one aminoacid for another amino acid that has similar properties.

Exemplary conservative substitutions are set out in Tables 1, 2, and 3below.

TABLE 1 Conservative Substitutions I SIDE-CHAIN CHARACTERISTIC AMINOACID Aliphatic Non-polar G A P I L V Polar - uncharged C S T M N QPolar - charged D E K R Aromatic H F W Y Other N Q D E

Alternatively, conservative amino acids can be grouped as described inLehninger, [Biochemistry, Second Edition; Worth Publishers, Inc. NY:NY(1975), pp. 71-77] as set out in Table 2, immediately below

TABLE 2 Conservative Substitutions II SIDE CHAIN CHARACTERISTIC AMINOACID Non-polar (hydrophobic) A. Aliphatic: A L I V P B. Aromatic: F W C.Sulfur-containing: M D. Borderline: G Uncharged-polar A. Hydroxyl: S T YB. Amides: N Q C. Sulfhydryl: C D. Borderline: G Positively Charged(Basic): K R H Negatively Charged (Acidic): D E

As still another alternative, exemplary conservative substitutions areset out in Table 3, immediately below.

TABLE 3 Conservative Substitutions III Original Residue ExemplarySubstitution Ala (A) Val, Leu, Ile Arg (R) Lys, Gln, Asn Asn (N) Gln,His, Lys, Arg Asp (D) Glu Cys (C) Ser Gln (Q) Asn Glu (E) Asp His (H)Asn, Gln, Lys, Arg Ile (I) Leu, Val, Met, Ala, Phe, Leu (L) Ile, Val,Met, Ala, Phe Lys (K) Arg, Gln, Asn Met (M) Leu, Phe, Ile Phe (F) Leu,Val, Ile, Ala Pro (P) Gly Ser (S) Thr Thr (T) Ser Trp (W) Tyr Tyr (Y)Trp, Phe, Thr, Ser Val (V) Ile, Leu, Met, Phe, Ala

Methods Directed to the Production of Virus and Host Cells of theInvention

The invention provides a method of modifying production of a virus thatis a member of the family Arteriviridae and Asfarviridae in a cellcomprising the step of directing said cell to express a CD163polypeptide. This may include rendering a virus non-permissive cell intoa virus permissive cell, or may involve rendering a cell more permissiveto the virus.

In one embodiment, the virus that is a member of the familyArteriviridae or Asfarviridae is selected from the group consisting ofLDV of mice, equine arteritis virus (EAV), simian hemorrhagic fevervirus (SHFV), PRRSV of swine, and ASFV of swine.

In a preferred embodiment the virus is PPRSV.

The invention further provides a method of preparing a culture of avirus that is a member of the family Arteriviridae or Asfarviridaecomprising the steps of: providing a cell line; directing said cell lineto express a CD163 polypeptide; infecting said cell line with virus; andcausing said cell line to produce viral progeny.

In one embodiment, the virus that is a member of the familyArteriviridae is selected from the group consisting of LDV of mice,equine arteritis virus (EAV), simian hemorrhagic fever virus (SHFV),PRRSV of swine and ASFV of swine.

In a preferred embodiment the virus is PRRSV.

All of the above methods utilize cells and cell lines expressing a CD163polypeptide. Expression of CD163 may be facilitated or increased bymethods that involve the introduction of exogenous nucleic acid into thecell. Such a cell may comprise a polynucleotide or vector in a mannerthat permits expression of an encoded CD163 polypeptide.

Polynucleotides that encode CD163 may be introduced into the host cellas part of a circular plasmid, or as linear DNA comprising an isolatedprotein-coding region, or in a viral vector. Methods for introducingexogenous nucleic acid into the host cell are well known and routinelypracticed in the art, including transformation, transfection,electroporation, nuclear injection, or fusion with carriers such asliposomes, micelles, ghost cells, and protoplasts. Host cell systems ofthe invention include invertebrate and vertebrate cells systems. Hostsmay include, but are not limited to, the following: insect cells,porcine kidney (PK) cells, feline kidney (FK) cells, swine testicular(ST) cells, African green monkey kidney cells (MA-104, MARC-145, VERO,and COS cells), Chinese hamster ovary (CHO) cells, baby hampster kidneycells, human 293 cells, and murine 3T3 fibroblasts. Insect host cellculture systems may also be used for the expression of CD163polypeptides. In another embodiment, the CD163 polypeptides areexpressed using a drosophila expression system.

The choice of a suitable expression vector for expression of the CD163polypeptides will of course depend upon the specific host cell to beused, and is within the skill of the ordinary artisan. Examples ofsuitable expression vectors include pSport and pcDNA3 (Invitrogen),pCMV-Script (Stratagene), and pSVL (Pharmacia Biotech). Expressionvectors for use in mammalian host cells may include transcriptional andtranslational control sequences derived from viral genomes. Commonlyused promoter sequences and modifier sequences which may be used in thepresent invention include, but are not limited to, those derived fromhuman cytomegalovirus (CMV), Rous sarcoma virus (RSV), Adenovirus 2,Polyoma virus, and Simian virus 40 (SV40). Methods for the constructionof mammalian expression vectors are disclosed, for example, in Okayamaand Berg (Mol. Cell. Biol. 3:280 (1983)); Cosman et al. (Mol. Immunol.23:935 (1986)); Cosman et al. (Nature 312:768 (1984)); EP-A-0367566; andWO 91/18982.

Because CD163 sequences are known to exist in cells from variousspecies, the endogenous gene may be modified to permit, or increase,expression of the CD163 polypeptide. Cells can be modified (e.g., byhomologous recombination) to provide increased expression by replacing,in whole or in part, the naturally occurring CD163 promoter with all orpart of a heterologous promoter, so that the cells express CD163polypeptide at higher levels. The heterologous promoter is inserted insuch a manner that it is operatively linked to endogenous CD163 encodingsequences. [See, for example, PCT International Publication No. WO94/12650, PCT International Publication No. WO 92/20808, and PCTInternational Publication No. WO 91/09955.] It is also contemplatedthat, in addition to heterologous promoter DNA, amplifiable marker DNA(e.g., ada, dhfr, and the multifunctional cad gene, which encodes forcarbamyl phosphate synthase, aspartate transcarbamylase, anddihydroorotase) and/or intron DNA may be inserted along with theheterologous promoter DNA. If linked to the CD163 coding sequence,amplification of the marker DNA by standard selection methods results inco-amplification of the CD163 coding sequences in the cells.

CD163 expression may also be induced by chemical treatment. Phorbolesters, especially phorbol myristyl acetate (PMA), activate one or moreisozymes of the ubiquitous membrane receptor, protein kinase C (PKC) andare particularly preferred means of increasing CD163 expresssion. Othermethods of intracellular calcium mobilization are also contemplated.

Vaccine Production

The methods described above may be used to produce any virus that is amember of the family Arteriviridae or Asfarviridae for the purpose ofvaccine production or diagnostics.

In one embodiment the virus that is a member of the family Arteriviridaeis selected from the group consisting of LDV of mice, equine arteritisvirus (EAV), simian hemorrhagic fever virus (SHFV), and PRRSV of swine.

In a preferred embodiment the virus is PRRSV.

Vaccine Production

The methods described above may be used to produce virus for the purposeof vaccine production or diagnostics.

Killed (inactivated) or live vaccines can be produced. Therefore, tomake a live vaccine, a viral isolate, or an attenuated or imitatedvariant thereof, is grown in cell culture. The virus is harvestedaccording to methods well known in the art. The virus may then beconcentrated, frozen, and stored at −70° C., or freeze-dried and storedat 4° C. Prior to vaccination the virus is mixed at an appropriatedosage, (which is from about 10³ to 10⁸ tissue culture infectious dosesper ml (TCID₅₀/ml)), with a pharmaceutically acceptable carrier such asa saline solution, and optionally an adjuvant.

The vaccine produced might also comprise an inactivated or killed viruscomprising a virus grown by the methods of the invention. Theinactivated vaccine is made by methods well known in the art. Forexample, once the virus is propagated to high titers, it would bereadily apparent by those skilled in the art that the virus antigenicmass could be obtained by methods well known in the art. For example,the virus antigenic mass may be obtained by dilution, concentration, orextraction. All of these methods have been employed to obtainappropriate viral antigenic mass to produce vaccines. The virus is theninactivated by treatment with formalin, betapropriolactone (BPL), binaryethyleneimine (BEI), or other methods known to those skilled in the art.The inactivated virus is then mixed with a pharmaceutically acceptablecarrier such as a saline solution, and optionally an adjuvant. Examplesof adjuvants include, but not limited to, aluminum hydroxide,oil-in-water and water-in-oil emulsions, AMPHIGEN, saponins such asQuilA, and polypeptide adjuvants including interleukins, interferons,and other cytokines.

Inactivation by formalin is performed by mixing the viral suspensionwith 37% formaldehyde to a final formaldehyde concentration of 0.05%.The virus-formaldehyde mixture is mixed by constant stirring forapproximately 24 hours at room temperature. The inactivated virusmixture is then tested for residual live virus by assaying for growth ona suitable cell line.

Inactivation by BEI is performed by mixing the viral suspension of thepresent invention with 0.1 M BEI (2-bromo-ethylamine in 0.175 N NaOH) toa final BEI concentration of 1 mM. The virus-BEI mixture is mixed byconstant stirring for approximately 48 hours at room temperature,followed by the addition of 1.0 M sodium thiosulfate to a finalconcentration of 0.1 mM. Mixing is continued for an additional twohours. The inactivated virus mixture is tested for residual live virusby assaying for growth on a suitable cell line.

Virus permissive cells that have been directed to express CD163 can alsobe used to quantify live virus. Two common methods, which are well knownto those skilled in the art, are the plaque assay and the limitingdilution assay.

CD163-expressing cell lines of the present invention can be used to growvirus for the purpose of producing viral antigen for diagnostic kits.For example lysates from infected cells (with optional purification ofviral particles or extraction of selected viral proteins) may be coatedon ELISA plates in order to detect and quantify antibodies to the virusin swine sera.

Live or inactivated virus grown in CD163-expressing cells can be usedafter optional separation of the viral proteins to immunize animals inorder to generate polyclonal, monospecific or monoclonal antibodies.These in turn can be used as the basis of diagnostic assays for thedetection and quantification of virus in swine serum and otherbiological samples.

Assays of the Invention

The invention provides methods for determining the propensity of ananimal to be infected by a virus that is a member of the familyArteriviridae or Asfarviridae, or of a cell line to support thereplication of a virus that is a member of the family Arteriviridae orAsfarviridae. Samples from either source are obtained and assayed forexpression of CD163. The level of CD163 gene expression can be comparedwith levels of controls known not to support replication of the virus,where an increased amount of CD163 gene expression relative to levelsfrom the controls indicates a greater propensity of the non-control tobe infected by said virus.

In the case of an animal, samples can be any sample comprising samplenucleic acid molecules or proteins, and obtained from any bodily tissueexpressing

CD163, including, but not limited to, alveolar macrophages, culturedcells, biopsies, or other tissue preparations. The level of expressioncan be assessed at the level of genomic DNA, messenger RNA, and/orprotein produced. In a preferred embodiment the member of the virus ofthe family Arteriviridae or Asfarviridae is selected from the groupconsisting of LDV of mice, equine arteritis virus (EAV), simianhemorrhagic fever virus (SHFV), PRRSV of swine, and ASFV of swine.

Nucleic Acid based Assays

Methods of determining CD163 levels may be nucleic acid based as notedabove. CD163-derived nucleic acids may be in solution or on a solidsupport. In some embodiments, they may be employed as array elements inmicroarrays alone or in combination with other array element molecules.Nucleic acid based methods generally require the isolation of DNA or RNAfrom the sample and subsequent hybridization or PCR amplification usingsprecific primers derived from any known CD163 encoding sequences in theart or those specifically disclosed as SEQ ID NOs: 1, 3, 5, 12, 13, 17,18, 22, 23, 25, 26, 30, 31, 33, 35, 37, 39, 41, 43, 45, and 47. DNA orRNA can be isolated from the sample according to any of a number ofmethods well known to those of skill in the art. For example, methods ofpurification of nucleic acids are described in Tijssen, P. (1993)Laboratory Techniques in Biochemistry and Molecular BiologyHybridization With Nucleic Acid Probes, Part I. Theory and Nucleic AcidPreparation, Elsevier, New York, N.Y. In one preferred embodiment, totalRNA is isolated using the TRIZOL total RNA isolation reagent (LifeTechnologies, Inc., Gaithersburg Md.) and mRNA is isolated using oligod(T) column chromatography or glass beads. When sample nucleic acidmolecules are amplified it is desirable to amplify the sample nucleicacid molecules and maintain the relative abundances of the originalsample, including low abundance transcripts. RNA can be amplified invitro, in situ, or in vivo (See Eberwine U.S. Pat. No. 5,514,545).

It is also advantageous to include controls within the sample to assurethat amplification and labeling procedures do not change the truedistribution of nucleic acid molecules in a sample. For this purpose, asample is spiked with an amount of a control nucleic acid moleculepredetermined to be detectable upon hybridization to its complementaryarrayed nucleic acid molecule and the composition of nucleic acidmolecules includes reference nucleic acid molecules which specificallyhybridize with the control arrayed nucleic acid molecules. Afterhybridization and processing, the hybridization signals obtained shouldreflect accurately the amounts of control arrayed nucleic acid moleculesadded to the sample.

Prior to hybridization, it may be desirable to fragment the samplenucleic acid molecules. Fragmentation improves hybridization byminimizing secondary structure and cross-hybridization to other samplenucleic acid molecules in the sample or noncomplementary nucleic acidmolecules. Fragmentation can be performed by mechanical or chemicalmeans.

Labeling

The sample nucleic acid molecules or probes may be labeled with one ormore labeling moieties to allow for detection of hybridizedarrayed/sample nucleic acid molecule complexes. The labeling moietiescan include compositions that can be detected by spectroscopic,photochemical, biochemical, bioelectronic, immunochemical, electrical,optical, or chemical means. The labeling moieties include radioisotopes,such as (32)P, (33)P or (35)S, chemiluminescent compounds, labeledbinding proteins, heavy metal atoms, spectroscopic markers, such asfluorescent markers and dyes, magnetic labels, linked enzymes, massspectrometry tags, spin labels, electron transfer donors and acceptors,and the like. Preferred fluorescent markers include Cy3 and Cy5fluorophores (Amersham Pharmacia Biotech, Piscataway N.J.).

Hybridization

The nucleic acid molecule sequence of SEQ ID NOs: 1, 3, 5, 12, 13, 17,18, 22, 23, 25, 26, 30, 31, 33, 35, 37, 39, 41, 43, 45, and 47 or otherCD163 encoding sequences in the art and fragments thereof can be used invarious hybridization technologies for various purposes. Hybridizationprobes may be designed or derived from any mammalian CD163 sequence butmay make use of those sequences disclosed in SEQ ID NOs: 1, 3, 5, 12,13, 17, 18, 22, 23, 25, 26, 30, 31, 33, 35, 37, 39, 41, 43, 45, and 47.Such probes may be made from a highly specific region or from aconserved motif, and used in protocols to quantify CD163 message,allelic variants, or related sequences. The hybridization probes of thesubject invention may be DNA or RNA and may be derived from anymammalian CD163 sequence known in the art or from those sequencesdisclosed herein as SEQ ID NOs: 1, 3, 5, 12, 13, 17, 18, 22, 23, 25, 26,30, 31, 33, 35, 37, 39, 41, 43, 45, and 47 or from genomic sequencesincluding promoters, enhancers, and introns of the mammalian gene.Hybridization or PCR probes may be produced using oligolabeling, nicktranslation, end-labeling, or PCR amplification in the presence of thelabeled nucleotide. A vector containing the nucleic acid sequence may beused to produce an mRNA probe in vitro by addition of an RNA polymeraseand labeled nucleic acid molecules. These procedures may be conductedusing commercially available kits such as those provided by AmershamPharmacia Biotech.

The stringency of hybridization is determined by the G+C content of theprobe, salt concentration, and temperature. In particular, stringencycan be increased by reducing the concentration of salt or raising thehybridization temperature. In solutions used for some membrane-basedhybridizations, additions of an organic solvent such as formamide allowsthe reaction to occur at a lower temperature. Hybridization can beperformed at low stringency with buffers, such as 5× saline sodiumcitrate (SSC) with 1% sodium dodecyl sulfate (SDS) at 60° C., whichpermits the formation of a hybridization complex between nucleotidesequences that contain some mismatches. Subsequent washes are performedat higher stringency with buffers such as 0.2×SSC with 0.1% SDS ateither 45° C. (medium stringency) or 68° C. (high stringency). At highstringency, hybridization complexes will remain stable only where thenucleic acid sequences are almost completely complementary. In somemembrane-based hybridizations, preferably 35% or most preferably 50%,formamide can be added to the hybridization solution to reduce thetemperature at which hybridization is performed, and background signalscan be reduced by the use of other detergents such as Sarkosyl or TritonX-100 and a blocking agent such as salmon sperm DNA. Selection ofcomponents and conditions for hybridization are well known to thoseskilled in the art and are reviewed in Ausubel (supra) and Sambrook etal. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring HarborPress, Plainview N.Y.

Exemplary highly stringent hybridization conditions are as follows:hybridization at 42° C. in a hybridization solution comprising 50%formamide, 1% SDS, 1M NaCl, 10% Dextran sulfate, and washing twice for30 minutes at 60° C. in a wash solution comprising 0.1×SSC and 1% SDS.It is understood in the art that conditions of equivalent stringency canbe achieved through variation of temperature and buffer, or saltconcentration as described in Ausubel, et al. (Eds.), Protocols inMolecular Biology, John Wiley & Sons (1994), pp. 6.0.3 to 6.4.10.Modifications in hybridization conditions can be empirically determinedor precisely calculated based on the length and the percentage ofguanosine/cytosine (GC) base pairing of the probe. The hybridizationconditions can be calculated as described in Sambrook, et al., (Eds.),Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress: Cold Spring Harbor, N.Y. (1989), pp. 9.47 to 9.51.

Hybridization specificity can be evaluated by comparing thehybridization of specificity-control nucleic acid molecules tospecificity-control sample nucleic acid molecules that are added to asample in a known amount. The specificity-control arrayed nucleic acidmolecules may have one or more sequence mismatches compared with thecorresponding arrayed nucleic acid molecules. In this manner, it ispossible to determine whether only complementary arrayed nucleic acidmolecules are hybridizing to the sample nucleic acid molecules orwhether mismatched hybrid duplexes are forming is determined.

Hybridization reactions can be performed in absolute or differentialhybridization formats. In the absolute hybridization format, nucleicacid molecules from one sample are hybridized to the molecules in amicroarray format and signals detected after hybridization complexformation correlate to nucleic acid molecule levels in a sample. In thedifferential hybridization format, the differential expression of a setof genes in two biological samples is analyzed. For differentialhybridization, nucleic acid molecules from both biological samples areprepared and labeled with different labeling moieties. A mixture of thetwo labeled nucleic acid molecules is added to a microarray. Themicroarray is then examined under conditions in which the emissions fromthe two different labels are individually detectable. Molecules in themicroarray that are hybridized to substantially equal numbers of nucleicacid molecules derived from both biological samples give a distinctcombined fluorescence (Shalon et al.; PCT publication WO95/35505). In apreferred embodiment, the labels are fluorescent markers withdistinguishable emission spectra, such as Cy3 and Cy5 fluorophores.

After hybridization, the microarray is washed to remove nonhybridizednucleic acid molecules and complex formation between the hybridizablearray elements and the nucleic acid molecules is detected. Methods fordetecting complex formation are well known to those skilled in the art.In a preferred embodiment, the nucleic acid molecules are labeled with afluorescent label and measurement of levels and patterns of fluorescenceindicative of complex formation is accomplished by fluorescencemicroscopy, preferably confocal fluorescence microscopy.

In a differential hybridization experiment, nucleic acid molecules fromtwo or more different biological samples are labeled with two or moredifferent fluorescent labels with different emission wavelengths.Fluorescent signals are detected separately with differentphotomultipliers set to detect specific wavelengths. The relativeabundances/expression levels of the nucleic acid molecules in two ormore samples are obtained.

Typically, microarray fluorescence intensities can be normalized to takeinto account variations in hybridization intensities when more than onemicroarray is used under similar test conditions. In a preferredembodiment, individual arrayed-sample nucleic acid molecule complexhybridization intensities are normalized using the intensities derivedfrom internal normalization controls contained on each microarray.

Polypeptide Based Assays

The present invention provides methods and reagents for detecting andquantifying CD163 polypeptides. These methods include analyticalbiochemical methods such as electrophoresis, mass spectroscopy,chromatographic methods and the like, or various immunological methodssuch as radioimmunoassay (RIA), enzyme-linked immunosorbent assays(ELISAs), immunofluorescent assays, western blotting, affinity capturemass spectrometry, biological activity, and others described below andapparent to those of skill in the art upon review of this disclosure.

Immunoassays

The present invention also provides methods for detection of CD163polypeptides employing one or more anti-CD163 antibody reagents (i.e.,immunoassays). As used herein, an immunoassay is an assay that utilizesan antibody (as broadly defined herein and specifically includesfragments, chimeras and other binding agents) that specifically binds aCD163 polypeptide or epitope.

A number of well-established immunological binding assay formatssuitable for the practice of the invention are known (see, e.g., U.S.Pat. Nos. 4,366,241; 4,376,110; 4,517,288; and 4,837,168). See, e.g.,Methods in Cell Biology Volume 37: Antibodies in Cell Biology, Asai, ed.Academic Press, Inc. New York (1993); Basic and Clinical Immunology 7thEdition, Stites & Terr, eds. (1991); Harlow and Lane, supra [e.g.,Chapter 14], and Ausubel et al., supra, [e.g., Chapter 11]. Typically,immunological binding assays (or immunoassays) utilize a “capture agent”to specifically bind to and, often, immobilize the analyte to a solidphase. In one embodiment, the capture agent is a moiety thatspecifically binds to a CD163 polypeptide or subsequence, such as ananti-CD163 antibody.

Usually the CD163 gene product being assayed is detected directly orindirectly using a detectable label. The particular label or detectablegroup used in the assay is usually not a critical aspect of theinvention, so long as it does not significantly interfere with thespecific binding of the antibody or antibodies used in the assay. Thelabel may be covalently attached to the capture agent (e.g., ananti-CD163 antibody), or may be attached to a third moiety, such asanother antibody, that specifically binds to the CD163 polypeptide.

The present invention provides methods and reagents for competitive andnoncompetitive immunoassays for detecting CD163 polypeptides.Noncompetitive immunoassays are assays in which the amount of capturedanalyte (in this case CD163) is directly measured. One such assay is atwo-site, monoclonal-based immunoassay utilizing monoclonal antibodiesreactive to two non-interfering epitopes on the CD163 polypeptide. See,e.g., Maddox et al., 1983, J. Exp. Med., 158:1211 for backgroundinformation. In one “sandwich” assay, the capture agent (e.g., ananti-CD163 antibody) is bound directly to a solid substrate where it isimmobilized. These immobilized antibodies then capture any CD163polypeptide present in the test sample. The CD163 polypeptide thusimmobilized can then be labeled, e.g., by binding to a second anti-CD163antibody bearing a label. Alternatively, the second CD163 antibody maylack a label, but be bound by a labeled third antibody specific toantibodies of the species from which the second antibody is derived. Thesecond antibody alternatively can be modified with a detectable moiety,such as biotin, to which a third labeled molecule can specifically bind,such as enzyme-labeled streptavidin.

In competitive assays, the amount of CD163 polypeptide present in thesample is measured indirectly by measuring the amount of an added(exogenous) CD163 polypeptide displaced (or competed away) from acapture agent (e.g., CD163 antibody) by the CD163 polypeptide present inthe sample. A hapten inhibition assay is another example of acompetitive assay. In this assay CD163 polypeptide is immobilized on asolid substrate. A known amount of CD163 antibody is added to thesample, and the sample is then contacted with the immobilized CD163polypeptide. In this case, the amount of anti-CD163 antibody bound tothe immobilized CD163 polypeptide is inversely proportional to theamount of CD163 polypeptide present in the sample. The amount ofimmobilized antibody may be detected by detecting either the immobilizedfraction of antibody or the fraction of the antibody that remains insolution. In this aspect, detection may be direct, where the antibody islabeled, or indirect where the label is bound to a molecule thatspecifically binds to the antibody as described above.

Other Antibody-Based Assay Formats

The invention also provides reagents and methods for detecting andquantifying the presence of CD163 polypeptide in the sample by using animmunoblot (Western blot) format. Another immunoassay is the so-called“lateral flow chromatography.” In a non-competitive version of lateralflow chromatography, a sample moves across a substrate by, e.g.,capillary action, and encounters a mobile-labeled antibody that bindsthe analyte forming a conjugate. The conjugate then moves across thesubstrate and encounters an immobilized second antibody that binds theanalyte. Thus, immobilized analyte is detected by detecting the labeledantibody. In a competitive version of lateral flow chromatography alabeled version of the analyte moves across the carrier and competeswith unlabeled analyte for binding with the immobilized antibody. Thegreater the amount of the analyte in the sample, the less the binding bylabeled analyte and, therefore, the weaker the signal. See, e.g., May etal., U.S. Pat. No. 5,622,871; and Rosenstein, U.S. Pat. No. 5,591,645.

Depending upon the assay, various components, including the antigen,target antibody, or anti-cathepsin S antibody, may be bound to a solidsurface or support (i.e., a substrate, membrane, or filter paper). Manymethods for immobilizing biomolecules to a variety of solid surfaces areknown in the art. For instance, the solid surface may be a membrane(e.g., nitrocellulose), a microtiter dish (e.g., PVC, polypropylene, orpolystyrene), a test tube (glass or plastic), a dipstick (e.g. glass,PVC, polypropylene, polystyrene, latex, and the like), a microcentrifugetube, or a glass or plastic bead. The desired component may becovalently bound or noncovalently attached through nonspecific bonding.

A wide variety of organic and inorganic polymers, both natural andsynthetic may be employed as the material for the solid surface.Illustrative polymers include polyethylene, polypropylene, poly(4-methylbutene), polystyrene, polymethacrylate, poly(ethyleneterephthalate), rayon, nylon, poly(vinylbutyrate), polyvinylidenedifluoride (PVDF), silicones, polyformaldehyde, cellulose, celluloseacetate, nitrocellulose, and the like. Other materials that may beemployed, include paper, glasses, ceramics, metals, metalloids,semiconductive materials, cements or the like. In addition, substancesthat form gels, such as proteins (e.g., gelatins), lipopolysaccharides,silicates, agarose and polyacrylamides can be used. Polymers that formseveral aqueous phases, such as dextrans, polyalkylene glycols orsurfactants, such as phospholipids, long chain (12-24 carbon atoms)alkyl ammonium salts and the like are also suitable. Where the solidsurface is porous, various pore sizes may be employed depending upon thenature of the system.

Mass Spectrometry

The mass of a molecule frequently can be used as an identifier of themolecule. Therefore, methods of mass spectrometry can be used toidentify a protein analyte. Mass spectrometers can measure mass bydetermining the time required for an ionized analyte to travel down aflight tube and to be detected by an ion detector. One method of massspectrometry for proteins is matrix-assisted laser desorption ionizationmass spectrometry (“MALDI”). In MALDI the analyte is mixed with anenergy absorbing matrix material that absorbs energy of the wavelengthof a laser and placed on the surface of a probe. Upon striking thematrix with the laser, the analyte is desorbed from the probe surface,ionized, and detected by the ion detector. See, for example, Hillenkampet al., U.S. Pat. No. 5,118,937.

Other methods of mass spectrometry for proteins are described inHutchens and Yip, U.S. Pat. No. 5,719,060. In one such method referredto as Surfaces Enhanced for Affinity Capture (“SEAC”) a solid phaseaffinity reagent that binds the analyte specifically ornon-specifically, such as an antibody or a metal ion, is used toseparate the analyte from other materials in a sample. Then the capturedanalyte is desorbed from the solid phase by, e.g., laser energy,ionized, and detected by the detector.

Nucleic Acids of the Invention

The examples below disclose our discovery of several novel CD163polynucleotides. The invention includes these novel CD163polynucleotides. The present invention provides several isolated novelpolynucleotides (e.g., DNA sequences and RNA transcripts, both sense andcomplementary antisense strands, both single and double-stranded,including splice variants thereof, which encode novel CD163polypeptides. We report herein isolated novel polynucleotides whichencode porcine, murine, human, canine, and african green monkey CD163polypeptides and which comprise the sequences set forth in SEQ ID NOs:1, 5, 12, 13, 22, 23, 25, 26, 30, 31, 33, 35, 37, 39, 41, 43, 45, and47.

It should be recognized that by disclosing SEQ ID NOs: 1, 5, 12, 13, 22,23, 25, 26, 30, 31, 33, 35, 37, 39, 41, 43, 45, and 47 it provides oneskilled in the art a multitude of methods of obtaining these sequences.By way of example, it would be possible to generate probes from thesequences disclosed in SEQ ID NOs: 1, 5, 12, 13, 22, 23, 25, 26, 30, 31,33, 35, 37, 39, 41, 43, 45, and 47 and screen porcine, murine, human,canine, and african green monkey cDNA or genomic libraries and therebyobtain the entire sequence disclosed in SEQ ID NOs: 1, 3, 5, 12, 13, 17,18, 22, 23, 25, 26, 30, 31, 33, 35, 37, 39, 41, 43, 45 and 47, or itsgenomic equivalent. Sambrook, et al., (Eds.), Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press: Cold SpringHarbor, N.Y. (1989). Also by way of example, one skilled in the artwould immediately recognize that given the sequence disclosed in SEQ IDNO: 1, 5, 12, 13, 22, 23, 25, 26, 30, 31, 33, 35, 37, 39, 41, 43, 45,and 47 it is then possible to generate the appropriate primers for PCRamplification to obtain the entire sequence represented by thesesequences. (See e.g., PCR Technology, H. A. Erlich, ed., Stockton Press,New York, 1989; PCR Protocols: A Guide to Methods and Applications, M.A. Innis, David H. Gelfand, John J. Sninsky, and Thomas J. White, eds.,Academic Press, Inc., New York, 1990.)

DNA polynucleotides of the invention include cDNA, and DNA that has beenchemically synthesized in whole or in part and is also intended toinclude allelic variants thereof. Allelic variants are modified forms ofa wild type gene sequence, the modification resulting from recombinationduring chromosomal segregation, or exposure to conditions which giverise to genetic mutation. Allelic variants, like wild type genes, arenaturally occurring sequences (as opposed to non-naturally occurringvariants which arise from in vitro manipulation).

DNA sequences encoding the novel CD163 polypeptides are set forth in SEQID NOs: 1, 5, 12, 13, 22, 23, 25, 26, 30, 31, 33, 35, 37, 39, 41, 43,45, and 47. The worker of skill in the art will readily appreciate thatthe DNA of the invention comprises a double stranded molecule, forexample the molecule having the sequence set forth in SEQ ID NO: 1, 5,12, 13, 22, 23, 25, 26, 30, 31, 33, 35, 37, 39, 41, 43, 45, and 47 alongwith the complementary molecule (the “non-coding strand” or“complement”) having a sequence deducible from the sequences of SEQ IDNOs: 1, 5, 12, 13, 22, 23, 25, 26, 30, 31, 33, 35, 37, 39, 41, 43, 45,and 47 according to Watson-Crick base pairing rules for DNA. Alsocontemplated by the invention are other polynucleotides encoding for theporcine, murine, and african green monkey CD163 polypeptides of SEQ IDNOS: 2, 14, 24, 27, 32, 34, 36, 38, 40, 42, 44, 46, and 48 which differin sequence from the polynucleotide of SEQ ID NOs: 1, 3, 5, 12, 13, 17,18, 22, 23, 25, 26, 30, 31, 33, 35, 37, 39, 41, 43, 45, and 47 by virtueof the well-known degeneracy of the universal genetic code, as is wellknown in the art. The present invention, therefore, contemplates thoseother DNA and RNA molecules that, on expression, encode the polypeptidesof SEQ ID NO: 2, 14, 24, 27, 32, 34, 36, 38, 40, 42, 44, 46, and 48.Having identified the amino acid residue sequence encoded the porcineCD163 polypeptide, and with the knowledge of all triplet codons for eachparticular amino acid residue, it is possible to describe all suchencoding RNA and DNA sequences. DNA and RNA molecules other than thosespecifically disclosed herein characterized simply by a change in acodon for a particular amino acid, are, therefore, within the scope ofthis invention.

A table of amino acids and their representative abbreviations, symbolsand codons is set forth below in the following Table 4.

TABLE 4 Amino acid Abbrev. Symbol Codon(s) Alanine Ala A GCA GCC GCG GCUCysteine Cys C UGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu EGAA GAG Phenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGUHistidine His H CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAAAAG Leucine Leu L UUA UUG CUA CUC CUG CUU Methionine Met M AUGAsparagine Asn N AAC AAU Proline Pro P CCA CCC CCG CCU Glutamine Gln QCAA CAG Arginine Arg R AGA AGG CGA CGC CGG CGU Serine Ser S AGC AGU UCAUCC UCG UCU Threonine Thr T ACA ACC ACG ACU Valine Val V GUA GUC GUG GUUTryptophan Trp W UGG Tyrosine Tyr Y UAC UAU

As is well known in the art, codons constitute triplet sequences ofnucleotides in mRNA and their corresponding cDNA molecules. Codons arecharacterized by the base uracil (U) when present in an mRNA moleculebut are characterized by the base thymidine (T) when present in DNA. Asimple change in a codon for the same amino acid residue within apolynucleotide will not change the sequence or structure of the encodedpolypeptide. It is apparent that when a phrase stating that a particular3 nucleotide sequence “encode(s)” any particular amino acid, theordinarily skilled artisan would recognize that the table above providesa means of identifying the particular nucleotides at issue. By way ofexample, if a particular three-nucleotide sequence encodes theonine thetable above discloses that the posible triplet sequences are ACA, ACG,ACC, and ACU (ACT if in DNA).

The invention includes therefore, an isolated polynucleotide comprising:

(a) a susCD163v1 polynucleotide sequence set forth in SEQ ID NOs: 1 and5;(b) a polynucleotide that encodes a polypeptide that has at least 70%,71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identity and/or similarity to a polypeptide set forth in SEQ ID NO:2;(c) a polynucleotide encoding a polypeptide of SEQ ID NO: 2;(d) a polynucleotide that is the complement of any of (a), (b), or (c).

The invention also includes an isolated polynucleotide comprising:

(a) a susCD163v2 polynucleotide sequence set forth in SEQ ID NOs: 12 or13;(b) a polynucleotide that encodes a polypeptide that has at least 99%identity and/or similarity to a polypeptide set forth in SEQ ID NO: 14;(c) a polynucleotide encoding a polypeptide of SEQ ID NO: 14;(d) a polynucleotide that is the complement of any of (a), (b), or (c).

The invention also includes an isolated polynucleotide comprising:

(a) a susCD163v2 polynucleotide sequence set forth in SEQ ID NOs: 17 or18;(b) a polynucleotide encoding a polypeptide of SEQ ID NO: 19;(d) a polynucleotide that is the complement of any of (a) or (b).

The invention also includes an isolated polynucleotide comprising:

(a) a CD63v2 polynucleotide sequence set forth in SEQ ID NOs: 22 or 23;(b) a polynucleotide encoding a polypeptide of SEQ ID NO: 24;(c) a polynucleotide that is the complement of (a) or (b).

The invention also includes an isolated polynucleotide comprising:

(a) a CD163v3 polynucleotide sequence set forth in SEQ ID NOs: 25 or 26;(b) a polynucleotide that encodes a polypeptide that has at least 96%,97%, 98%, or 99% identity and/or similarity to a polypeptide set forthin SEQ ID NO: 27;(c) a polynucleotide encoding a polypeptide of SEQ ID NO: 27;(d) a polynucleotide that is the complement of any of (a), (b), or (c).

The invention also includes an isolated polynucleotide comprising:

(a) a CD163v2 polynucleotide sequence set forth in SEQ ID NOs: 30 or 31;(b) a polynucleotide that encodes a polypeptide that has at least 98% or99% identity and/or similarity to a polypeptide set forth in SEQ ID NO:32;(c) a polynucleotide encoding a polypeptide of SEQ ID NO: 32;(d) a polynucleotide that is the complement of any of (a), (b), or (c).

The invention also includes an isolated polynucleotide comprising:

(a) a polynucleotide sequence set forth in SEQ ID NO: 33;(b) a polynucleotide that encodes a polypeptide that has at least 95%,96%, 97%, 98%, or 99% identity and/or similarity to a polypeptide setforth in SEQ ID NO: 34;(c) a polynucleotide encoding a polypeptide of SEQ ID NO: 34;(d) a polynucleotide that is the complement of any of (a), (b), or (c).

The invention also includes an isolated polynucleotide comprising:

(a) a polynucleotide sequence set forth in SEQ ID NO: 35;(b) a polynucleotide that encodes a polypeptide that has at least 95%,96%, 97%, 98%, or 99% identity and/or similarity to a polypeptide setforth in SEQ ID NO: 36;(c) a polynucleotide encoding a polypeptide of SEQ ID NO: 36;(d) a polynucleotide that is the complement of any of (a), (b), or (c).

The invention also includes an isolated polynucleotide comprising:

(a) a polynucleotide sequence set forth in SEQ ID NO: 37;(b) a polynucleotide that encodes a polypeptide that has at least 95%,96%, 97%, 98%, or 99% identity and/or similarity to a polypeptide setforth in SEQ ID NO: 38(c) a polynucleotide encoding a polypeptide of SEQ ID NO: 38;(d) a polynucleotide that is the complement of any of (a), (b), or (c).

The invention also includes an isolated polynucleotide comprising:

(a) a polynucleotide sequence set forth in SEQ ID NO: 39;(b) a polynucleotide that encodes a polypeptide that has at least 95%,96%, 97%, 98%, or 99% identity and/or similarity to a polypeptide setforth in SEQ ID NO: 40;(c) a polynucleotide encoding a polypeptide of SEQ ID NO: 40;(d) a polynucleotide that is the complement of any of (a), (b), or (c).

The invention also includes an isolated polynucleotide comprising:

(a) a polynucleotide sequence set forth in SEQ ID NO: 41;(b) a polynucleotide that encodes a polypeptide that has at least 95%,96%, 97%, 98%, or 99% identity and/or similarity to a polypeptide setforth in SEQ ID NO: 42;(c) a polynucleotide encoding a polypeptide of SEQ ID NO: 42;(d) a polynucleotide that is the complement of any of (a), (b), or (c).

The invention also includes an isolated polynucleotide comprising:

(a) a polynucleotide sequence set forth in SEQ ID NO: 43;(b) a polynucleotide that encodes a polypeptide that has at least 95%,96%, 97%, 98%, or 99% identity and/or similarity to a polypeptide setforth in SEQ ID NO: 44;(c) a polynucleotide encoding a polypeptide of SEQ ID NO: 44;(d) a polynucleotide that is the complement of any of (a), (b), or (c).

The invention also includes an isolated polynucleotide comprising:

(a) a polynucleotide sequence set forth in SEQ ID NO: 45;(b) a polynucleotide that encodes a polypeptide that has at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity and/orsimilarity to a polypeptide set forth in SEQ ID NO: 46;(c) a polynucleotide encoding a polypeptide of SEQ ID NO: 46;(d) a polynucleotide that is the complement of any of (a), (b), or (c).

The invention also includes an isolated polynucleotide comprising:

(a) a polynucleotide sequence set forth in SEQ ID NO: 47;(b) a polynucleotide that encodes a polypeptide that has at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity and/orsimilarity to a polypeptide set forth in SEQ ID NO: 48;(c) a polynucleotide encoding a polypeptide of SEQ ID NO: 49;(d) a polynucleotide that is the complement of any of (a), (b), or (c).

The polynucleotide sequence information provided by the invention makespossible large-scale expression of the encoded polypeptide by techniqueswell known and routinely practiced in the art. Polynucleotides of theinvention also permit identification and isolation of polynucleotidesencoding related porcine CD163v1 polypeptides, such as human allelicvariants and species homologs, by well-known techniques includingSouthern and/or Northern hybridization, and the polymerase chainreaction (PCR).

Knowledge of the sequence of any of the CD163 sequences disclosed hereinalso makes possible through use of Southern hybridization or polymerasechain reaction (PCR), the identification of genomic DNA sequencesencoding CD163 regulatory sequences, such as promoters, operators,enhancers, repressors, and the like.

As noted in the section above entitled “Assays of the Invention”polynucleotides of the invention are also useful in hybridization assaysto detect the capacity of cells to express CD163, or to measure levelsof CD163 expression. Polynucleotides of the invention may also be thebasis for diagnostic methods useful for determining the susceptibilityof an animal to virus infection as described above.

The disclosure herein of the full-length polynucleotides encoding aCD163 polypeptide makes readily available to the worker of ordinaryskill in the art fragments of the full length polynucleotide. Theinvention therefore provides unique fragments of the CD163 encodingpolynucleotides comprising at least 15 through the length of thefull-length sequence (including each and every integer value between)consecutive nucleotides of a polynucleotide encoding a CD163 disclosedherein. Because polynucleotides of the invention (including fragments)comprise sequences unique to the particular CD163-encodingpolynucleotide sequence, they therefore would hybridize under highlystringent or moderately stringent conditions only (i.e., “specifically”)to polynucleotides encoding the various CD163 polypeptides. Sequencesunique to polynucleotides of the invention are recognizable throughsequence comparison to other known polynucleotides, and can beidentified through use of alignment programs routinely utilized in theart, e.g., those made available in public sequence databases. Suchsequences also are recognizable from Southern hybridization analyses todetermine the number of fragments of genomic DNA to which apolynucleotide will hybridize. Polynucleotides of the invention can belabeled in a manner that permits their detection, including radioactive,fluorescent, and enzymatic labeling.

One or more unique fragment polynucleotides (or other CD163polynucleotides as discussed above) can be included in kits that areused to detect the presence of a polynucleotide encoding for CD163, orused to detect variations in a polynucleotide sequence encoding forCD163. Also made available by the invention are anti-sensepolynucleotides that recognize and hybridize to polynucleotides encodingCD163. Full length and fragment anti-sense polynucleotides are provided.Fragment anti-sense molecules of the invention include (i) those thatspecifically recognize and hybridize to the CD163 variants disclosedherein (as determined by sequence comparison of DNA encoding CD163s toDNA encoding other known molecules). Identification of sequences uniqueto the novel CD163-encoding polynucleotides can be deduced through theuse of any publicly available sequence database, and/or through the useof commercially available sequence comparison programs. The uniquenessof selected sequences in an entire genome can be further verified byhybridization analyses. After identification of the desired sequences,isolation through restriction digestion or amplification using any ofthe various polymerase chain reaction techniques well known in the artcan be performed. Anti-sense polynucleotides are particularly relevantto regulating expression of CD163 by those cells expressing CD163 mRNA.

Antisense nucleic acids (preferably 10 to 20 base pair oligonucleotides)capable of specifically binding to CD163 expression control sequences orCD163 RNA are introduced into cells (e.g., by a viral vector orcolloidal dispersion system such as a liposome). The antisense nucleicacid binds to the porcine CD163 target nucleotide sequence in the celland prevents transcription or translation of the target sequence.Phosphorothioate and methylphosphonate antisense oligonucleotides arespecifically contemplated for therapeutic use by the invention. Theantisense oligonucleotides may be further modified by poly-L-lysine,transferrin polylysine, or cholesterol moieties at their 5′ end.Suppression of porcine CD163 expression at either the transcriptional ortranslational level is useful to generate cellular or animal models fordiseases characterized by aberrant porcine CD163 expression or as atherapeutic modality.

As noted above in more detail, the nucleic acids of the inventioninclude vectors comprising a polynucleotide of the invention. Suchvectors are useful, e.g., for amplifying the polynucleotides in hostcells to create useful quantities thereof. In other embodiments, thevector is an expression vector wherein the polynucleotide of theinvention is operatively linked to a polynucleotide comprising anexpression control sequence. Such vectors are useful for recombinantproduction of polypeptides of the invention.

Also as noted above the invention provides host cells that aretransformed or transfected (stably or transiently) with polynucleotidesof the invention or vectors of the invention. As stated above, such hostcells are useful for the production of virus and the production ofvaccines.

The invention also provides isolated CD163 polypeptides encoded by anovel polynucleotide of the invention.

Polypeptides of the Invention

The examples disclose our discovery of several novel CD163 polypeptides.The invention includes these novel CD163 polypeptide which are set forthin SEQ ID NOs: 2, 14, 19, 24, 27, 32, 34, 36, 38, 40, 42, 44, 46, and48.

The invention includes therefore, an isolated polynucleotide comprisinga susCD163v1 polypetide with the sequence set forth in SEQ ID NO: 2.

The invention also includes a polypeptide that has at least 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity and/or similarity to a polypeptide set forth in SEQ ID NO: 2.

The invention includes therefore, an isolated polynucleotide comprisinga susCD163v2 polypetide with the sequence set forth in SEQ ID NO: 14.

The invention also includes a polypeptide that has at least 99%,identity and/or similarity to a sus CD163v2 polypeptide set forth in SEQID NO: 14.

The invention also includes a CD163v2 polypeptide having the sequenceset forth in SEQ ID NO: 24.

The invention also includes a CD163v3 polypeptide having the sequenceset forth in SEQ ID NO: 27.

The invention also includes a polypeptide having at least 96%, 97% 98%,or 99% identity and/or similarity to a polypeptide set forth in SEQ IDNO: 27.

The invention also includes a polypeptide having the sequence set forthin SEQ ID NO: 32.

The invention also includes a polypeptide that has at least 98% or 99%identity and/or similarity to a polypeptide set forth in SEQ ID NO: 32.

The invention also includes a polypeptide having the sequence set forthin SEQ ID NO: 34.

The invention also includes a polypeptide that has at least 95%, 96%,97%, 98%, or 99% identity and/or similarity to a polypeptide set forthin SEQ ID NO: 34.

The invention also includes a polypeptide having the sequence set forthin SEQ ID NO: 36.

The invention also includes a polypeptide that has at least 95%, 96%,97%, 98%, or 99% identity and/or similarity to a polypeptide set forthin SEQ ID NO: 36.

The invention also includes a polypeptide having the sequence set forthin SEQ ID NO: 38.

The invention also includes a polypeptide that has at least 95%, 96%,97%, 98%, or 99% identity and/or similarity to a polypeptide set forthin SEQ ID NO: 39.

The invention also includes a polypeptide having the sequence set forthin SEQ ID NO: 40.

The invention also includes a polypeptide that has at least 95%, 96%,97%, 98%, or 99% identity and/or similarity to a polypeptide set forthin SEQ ID NO: 40.

The invention also includes a polypeptide having the sequence set forthin SEQ ID NO: 42.

The invention also includes a polypeptide that has at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity and/or similarity toa polypeptide set forth in SEQ ID NO: 42.

The invention also includes a polypeptide having the sequence set forthin SEQ ID NO: 44.

The invention also includes a polypeptide that has at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity and/or similarity toa polypeptide set forth in SEQ ID NO: 44.

The invention also includes a polypeptide having the sequence set forthin SEQ ID NO: 46.

The invention also includes a polypeptide that has at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity and/or similarity toa polypeptide set forth in SEQ ID NO: 46.

The invention also includes a polypeptide having the sequence set forthin SEQ ID NO: 48.

The invention also includes a polypeptide that has at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity and/or similarity toa polypeptide set forth in SEQ ID NO: 48.

Polypeptides of the invention may be isolated from natural cell sourcesor may be chemically synthesized, but are preferably produced byrecombinant procedures involving host cells of the invention. Use ofmammalian host cells is expected to provide for such post-translationalmodifications (e.g., glycosylation, truncation, lipidation, andphosphorylation) as may be needed to confer optimal biological activityon recombinant expression products of the invention. Glycosylated andnon-glycosylated forms of the novel CD163 polypeptides are embraced.

Overexpression in eukaryotic and prokaryotic hosts as described abovefacilitates the isolation of CD163 polypeptides. The invention thereforeincludes isolated CD163 polypeptides as set out in SEQ ID NOs: 2, 14,19, 24, 27, 32, 34, 36, 38, 40, 42, 44, 46, and 48 and variants andconservative amino acid substitutions therein including labeled andtagged polypeptides.

The invention includes novel CD163 polypeptides that are “labeled”. Theterm “labeled” is used herein to refer to the conjugating or covalentbonding of any suitable detectable group, including enzymes (e.g.,horseradish peroxidase, beta-glucuronidase, alkaline phosphatase, andbeta-D-galactosidase), fluorescent labels (e.g., fluorescein,luciferase), and radiolabels (e.g., ¹⁴C, ¹²⁵I, ³H, ³²P, and ³⁵S) to thecompound being labeled. Techniques for labeling various compounds,including proteins, peptides, and antibodies, are well known. See, e.g.,Morrison, Methods in Enzymology 32b, 103 (1974); Syvanen et al., J.Biol. Chem. 284, 3762 (1973); Bolton and Hunter, Biochem. J. 133, 529(1973). The termed labeled may also encompass a polypeptide which hascovalently attached an amino acid tag as discussed below.

In addition, the novel CD163 polypeptides of the invention may beindirectly labeled. This involves the covalent addition of a moiety tothe polypeptide and subsequent coupling of the added moiety to a labelor labeled compound that exhibits specific binding to the added moiety.Possibilities for indirect labeling include hiotinylation of the peptidefollowed by binding to avidin coupled to one of the above label groups.Another example would be incubating a radiolabeled antibody specific fora histidine tag with a CD163s polypeptide comprising a polyhistidinetag. The net effect is to bind the radioactive antibody to thepolypeptide because of the considerable affinity of the antibody for thetag.

The invention also embraces variants (or analogs) of the novel CD163protein. In one example, insertion variants are provided wherein one ormore amino acid residues supplement a novel CD163 amino acid sequence.Insertions may be located at either or both termini of the protein, ormay be positioned within internal regions of the novel CD163 proteinamino acid sequence. Insertional variants with additional residues ateither or both termini can include for example, fusion proteins andproteins including amino acid tags or labels. Insertion variants includenovel CD163 polypeptides wherein one or more amino acid residues areadded to a CD163 acid sequence, or to a biologically active fragmentthereof.

Insertional variants therefore can also include fusion proteins whereinthe amino and/or carboxy termini of the novel CD163 polypeptide is fusedto another polypeptide. Various tag polypeptides and their respectiveantibodies are well known in the art. Examples include poly-histidine(poly-his) or poly-histidine-glycine (poly-his-gly) tags; the influenzaHA tag polypeptide and its antibody 12CA5 [Field et al., Mol. Cell.Biol., 8:2159-2165 (1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7and 9E10 antibodies thereto [Evan et al., Molecular and CellularBiology,5:3610-3616 (1985)], and the Herpes Simplex virus glycoprotein D (gD)tag and its antibody [Paborsky et al., Protein Engineering, 3(6):547-553(1990)]. Other tag polypeptides include the Flag-peptide [Hopp et al.,BioTechnology, 6:1204-1210 (1988)]; the KT3 epitope peptide [Martin etal., Science, 255:192-194 (1992)]; an alpha-tubulin epitope peptide[Skinner et al., J. Biol. Chem., 266:15163-15166 (1991)]; and the T7gene 10 protein peptide tag [Lutz-Freyermuth et al., Proc. Natl. Acad.Sci. USA, 87:6393-6397 (1990)]. In addition, the CD163 polypeptide canbe tagged with enzymatic proteins such as peroxidase and alkalinephosphatase.

In another aspect, the invention provides deletion variants wherein oneor more amino acid residues in a novel CD163 polypeptide is removed.Deletions can be effected at one or both termini of the novel CD163polypeptide, or with removal of one or more residues within the novelCD163 amino acid sequence. Deletion variants, therefore, include allfragments of the novel CD163 polypeptide.

CD163 polypeptides contain a transmembrane or membrane anchor region. Itshould be recognized that such transmembrane domains are useful whenexpressed in the context of a heterologous protein to aid in thetargeting of the heterologous protein to membranes. It should also berecognized that it may be advantageous to delete some transmembranedomains to enhance the purification or solubility of the protein.Transmembrane deleted variants of CD163 and polynucleotides encodingthem are of potential value as antiviral therapeutics. Such variants arespecifically disclosed here as SEQ ID NOs: 37-40.

The present invention also includes variants of the aforementionedpolypetides, that is, polypeptides that vary from the reference sequenceby conservative amino acid substitutions,

Exemplary conservative substitutions are set out in Tables 1, 2 and 3 inthe section above entitled “Definitions”.

In those situations where it is preferable to partially or completelyisolate the novel CD163 polypeptides, purification can be accomplishedusing standard methods well known to the skilled artisan. Such methodsinclude, without limitation, separation by electrophoresis followed byelectroelution, various types of chromatography (immunoaffinity,molecular sieve, and/or ion exchange), and/or high pressure liquidchromatography. In some cases, it may be preferable to use more than oneof these methods for complete purification.

Purification of novel CD163 polypeptides can be accomplished using avariety of techniques. If the polypeptide has been synthesized such thatit contains a tag such as Hexahistidine (CD163/hexaHis) or other smallpeptide such as FLAG (Eastman Kodak Co., New Haven, Conn.) or myc(Invitrogen, Carlsbad, Calif.) at either its carboxyl or amino terminus,it may essentially be purified in a one-step process by passing thesolution through an affinity column where the column matrix has a highaffinity for the tag or for the polypeptide directly (i.e., a monoclonalantibody specifically recognizing CD163). For example, polyhistidinebinds with great affinity and specificity to nickel, thus an affinitycolumn of nickel (such as the Qiagen Registered™ nickel columns) can beused for purification of CD163/polyHis. (See for example, Ausubel etal., eds., Current Protocols in Molecular Biology, Section 10.11.8, JohnWiley & Sons, New York [1993]).

Even if the novel CD163 polypeptide is prepared without a label or tagto facilitate purification, the novel CD163 of the invention may bepurified by immunoaffinity chromatography. To accomplish this,antibodies specific for CD163 polypeptides must be prepared by meanswell known in the art.

Antibodies generated against the novel CD163 polypeptides of theinvention can be obtained by administering the polypeptides orepitope-bearing fragments, analogues, or cells to an animal, preferablya non-human, using routine protocols. For preparation of monoclonalantibodies, any technique known in the art that provides antibodiesproduced by continuous cell line cultures can be used. Examples includevarious techniques, such as those in Kohler, G. and Milstein, C., Nature256: 495-497 (1975); Kozbor et al., Immunology Today 4: 72 (1983); Coleet al., pg. 77-96 in Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, Inc. (1985).

Where the novel CD163 polypeptides are prepared without a tag attached,and no antibodies are available, other well-known procedures forpurification can be used. Such procedures include, without limitation,ion exchange chromatography, molecular sieve chromatography, HPLC,native gel electrophoresis in combination with gel elution, andpreparative isoelectric focusing (“Isoprime” machine/technique, HoeferScientific). In some cases, two or more of these techniques may becombined to achieve increased purity.

It should be understood that the definition of polypeptides of theinvention is intended to include polypeptides bearing modificationsother than insertion, deletion, or substitution of amino acid residues.By way of example, the modifications may be covalent in nature, andinclude for example, chemical bonding with polymers, lipids, otherorganic, and inorganic moieties.

Antibodies

Also comprehended by the present invention are antibodies (e.g.,monoclonal and polyclonal antibodies, single chain antibodies, chimericantibodies, bifunctional/bispecific antibodies, humanized antibodies,human antibodies, and complementary determining region (CDR)-graftedantibodies, including compounds which include CDR sequences whichspecifically recognize a polypeptide of the invention) specific fornovel CD163 or fragments thereof.

The term “specific for,” when used to describe antibodies of theinvention, indicates that the variable regions of the antibodies of theinvention recognize and bind CD163s polypeptides exclusively (i.e., ableto distinguish CD163s polypeptides from other known polypeptides byvirtue of measurable differences in binding affinity, despite thepossible existence of localized sequence identity, homology, orsimilarity between the novel CD163 and such polypeptides). It will beunderstood that specific antibodies may also interact with otherproteins (for example, S. aureus protein A or other antibodies in ELISAtechniques) through interactions with sequences outside the variableregion of the antibodies, and in particular, in the constant region ofthe molecule. Screening assays to determine binding specificity of anantibody of the invention are well known and routinely practiced in theart. For a comprehensive discussion of such assays, see Harlow et al.(Eds), Antibodies A Laboratory Manual; Cold Spring Harbor Laboratory;Cold Spring Harbor, N.Y. (1988), Chapter 6. Antibodies that recognizeand bind fragments of the CD163s polypeptides of the invention are alsocontemplated, provided that the antibodies are, first and foremost,specific for novel CD163 polypeptides. Antibodies of the invention canbe produced using any method well known and routinely practiced in theart. Non-human antibodies may be humanized by any methods known in theart. In one method, the non-human CDRs are inserted into a humanantibody or consensus antibody framework sequence. Further changes canthen be introduced into the antibody framework to modulate affinity orimmunogenicity.

Antibodies of the invention are useful for, diagnostic purposes todetect or quantitate CD163s, as well as purification of CD163s. Kitscomprising an antibody of the invention for any of the purposesdescribed herein are also contemplated. In general, a kit of theinvention also includes a control antigen for which the antibody isimmunospecific

The present invention is further illustrated, but not limited, by thefollowing examples.

Example 1 Transient Transfection with Porcine CD163 Confers Permissivityto PRRS Virus Infection to a Non-Permissive Cell Line

Total mRNA from primary porcine alveolar macrophage cells was used toconstruct a cDNA library in the plasmid pCMV-Sport6.1 (Invitrogen), withthe cDNA cloned between the EcoRV and NotI sites. A member of thislibrary, when isolated and transiently transfected into the BHK-21 (babyhamster kidney) cell line, conferred a PRRS-permissive phenotype. Cellswere grown in Dulbecco's modified Eagle medium (DMEM) supplemented with5% fetal bovine serum (FBS) in a 5% CO₂ atmosphere at 37° C. Cellcultures were transiently transfected using 10.0 uL of Lipofectamine2000 (Invitrogen) and 2.0 ug of plasmid. A duplicate monolayer wastransfected with negative control plasmid pPAMB. This plasmid ispCMV-Sport6.1 lacking an insert. Transfection efficiency was monitoredwith a plasmid expressing green fluorescent protein (GFP). Approximately24 hours post-transfection, monolayers were infected with either NorthAmerican (isolate P129) or European (isolate 96V198) genotypes of PRRSvirus. For detection of PRRS replication, the monolayers were fixedusing 80% acetone approximately 24 hours post-infection and incubatedfor approximately 1 hour with FITC-conjugated monoclonal antibody SDOW17(Rural Technologies Inc.). This monoclonal antibody is specific for PRRSviral nucleocapsid expressed from open reading frame 7. A Nikon TE 300inverted fluorescent microscope with a 10× objective was used tophotograph a monolayer containing FITC positive cells and a negativecontrol monolayer.

It was confirmed that transfected cells became permissive to both theNorth American (isolate P129) and European (isolate 96V198) genotypes ofPRRSV. Expression of viral genes could be detected in many of thetransfected BHK cells, and progeny virus was readily detectable in thesupernatant. Control transfections using vector without insert orirrelevant plasmids did not confer permissivity.

Sequencing of the insert in the functional plasmid, using the Big DyeTerminator Version 1.0 Sequence Reaction kit (Applied Biosystems, FosterCity, Calif.) and the Applied Biosystems 3730 DNA Analyzer (AppliedBiosystems), revealed a gene that was highly homologous to the publishedporcine CD163 gene cDNA (Genbank accession number AJ311716). The cDNA weidentified contained additional 5′ and 3′ untranslated regions relativeto AJ311716, and the open reading frame differed in three ways: (1) a738 bp internal deletion near the 5′ end, (2) a 15 bp extension of the5′ end to an upstream ATG codon, and (3) sixteen nucleotide changespredicted to cause 10 amino acid changes. Nucleotide sequence identitybetween the sequences was 99.4%. Alignments of the newly discoveredporcine CD163 sequence with the previously reported sequence AJ311716are shown in FIGS. 1 and 2. The novel porcine CD163 variant wasdesignated “susCD163v1”.

SEQUENCE ID NO gtaataatac aagaagattt aaatgggcat aaaaccttgg aatggacaaactcagaatgg 60 SEQ ID tgctacatga aaactctgga tctgcagacc tgaaactgagagtggtagat ggagtcactg 120 NO: 5 aatgttcagg aagattggaa gtgaaattccaaggagaatg gggaacaatc tgtgatgatg 180 gctgggatag tgatgatgcc gctgtggcatgtaagcaact gggatgtcca actgctgtca 240 ctgccattgg tcgagttaac gccagtgagggaactggaca catttggctt gacagtgttt 300 cttgccatgg acacgagtct gctctctggcagtgtagaca ccatgaatgg ggaaagcatt 360 attgcaatca taatgaagat gctggtgtgacatgttctga tggatcagat ctggaactga 420 gacttaaagg tggaggcagc cactgtgctgggacagtgga ggtggaaatt cagaaactgg 480 taggaaaagt gtgtgataga agctggggactgaaagaagc tgatgtggtt tgcaggcagc 540 tgggatgtgg atctgcactc aaaacatcatatcaagttta ttccaaaacc aaggcaacaa 600 acacatggct gtttgtaagc agctgtaatggaaatgaaac ttctctttgg gactgcaaga 660 attggcagtg gggtggactt agttgtgatcactatgacga agccaaaatt acctgctcag 720 cccacaggaa acccaggctg gttggaggggacattccctg ctctggtcgt gttgaagtac 780 aacatggaga cacgtggggc accgtctgtgattctgactt ctctctggag gcggccagcg 840 tgctgtgcag ggaactacag tgcggcactgtggtttccct cctgggggga gctcactttg 900 gagaaggaag tggacagatc tgggctgaagaattccagtg tgaggggcac gagtcccacc 960 tttcactctg cccagtagca ccccgccctgacgggacatg tagccacagc agggacgtcg 1020 gcgtagtctg ctcaagatac acacaaatccgcttggtgaa tggcaagacc ccatgtgaag 1080 gaagagtgga gctcaacatt cttgggtcctgggggtccct ctgcaactct cactgggaca 1140 tggaagatgc ccatgtttta tgccagcagcttaaatgtgg agttgccctt tctatcccgg 1200 gaggagcacc ttttgggaaa ggaagtgagcaggtctggag gcacatgttt cactgcactg 1260 ggactgagaa gcacatggga gattgttccgtcactgctct gggcgcatca ctctgttctt 1320 cagggcaagt ggcctctgta atctgctcagggaaccagag tcagacacta tccccgtgca 1380 attcatcatc ctcggaccca tcaagctctattatttcaga agaaaatggt gttgcctgca 1440 tagggagtgg tcaacttcgc ctggtcgatggaggtggtcg ttgtgctggg agagtagagg 1500 tctatcatga gggctcctgg ggcaccatctgtgatgacag ctgggacctg aatgatgccc 1560 atgtggtgtg caaacagctg agctgtggatgggccattaa tgccactggt tctgctcatt 1620 ttggggaagg aacagggccc atttggctggatgagataaa ctgtaatgga aaagaatctc 1680 atatttggca atgccactca catggttgggggcggcacaa ttgcaggcat aaggaggatg 1740 caggagtcat ctgctcggag ttcatgtctctcagactgat cagtgaaaac agcagagaga 1800 cctgtgcagg gcgcctggaa gttttttacaacggagcttg gggcagcgtt ggcaagaata 1860 gcatgtctcc agccacagtg ggggtggtatgcaggcagct gggctgtgca gacagagggg 1920 acatcagccc tgcatcttca gacaagacagtgtccaggca catgtgggtg gacaatgttc 1980 agtgtcctaa aggacctgac accctatggcagtgcccatc atctccatgg aagaagagac 2040 tggccagccc ctcagaggag acatggatcacatgtgccaa caaaataaga cttcaagaag 2100 gaaacactaa ttgttctgga cgtgtggagatctggtacgg aggttcctgg ggcactgtgt 2160 gtgacgactc ctgggacctt gaagatgctcaggtggtgtg ccgacagctg ggctgtggct 2220 cagctttgga ggcaggaaaa gaggccgcatttggccaggg gactgggccc atatggctca 2280 atgaagtgaa gtgcaagggg aatgaaacctccttgtggga ttgtcctgcc agatcctggg 2340 gccacagtga ctgtggacac aaggaggatgctgctgtgac gtgttcagaa attgcaaaga 2400 gccgagaatc cctacatgcc acaggtcgctcatcttttgt tgcacttgca atctttgggg 2460 tcattctgtt ggcctgtctc atcgcattcctcatttggac tcagaagcga agacagaggc 2520 agcggctctc agttttctca ggaggagagaattctgtcca tcaaattcaa taccgggaga 2580 tgaattcttg cctgaaagca gatgaaacggatatgctaaa tccctcagga gaccactctg 2640 aagtacaatg aaaaggaaaa tgggaattataacctggtga gttcagcctt taagatacct 2700 tgatgaagac ctggactatt gaatgagcaagaatctgcct cttacactga agattacaat 2760 acagtcctct gtctcctggt attccaaagactgctgttga atttctaaaa aatagattgg 2820 tgaatgtgac tactcaaagt tgtatgtaagactttcaagg gcattaaata aaaaagaata 2880 ttgctgaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa 2930 1M  D  K  L  R  M  V  L  H  E  N  S  G  S  A  D  L  K  L  R SEQ ID 1atggacaaactcagaatggtgctacatgaaaactctggatctgcagacctgaaactgaga NO: 1 21V  V  D  G  V  T  E  C  S  G  R  L  E  V  K  F  Q  G  E  W and 2 61gtggtagatggagtcactgaatgttcaggaagattggaagtgaaattccaaggagaatgg 41G  T  I  C  D  D  G  W  D  S  D  D  A  A  V  A  C  K  Q  L 121ggaacaatctgtgatgatggctgggatagtgatgatgccgctgtggcatgtaagcaactg 61G  C  P  T  A  V  T  A  I  G  R  V  N  A  S  E  G  T  G  H 181ggatgtccaactgctgtcactgccattggtcgagttaacgccagtgagggaactggacac 81I  W  L  D  S  V  S  C  H  G  H  E  S  A  L  W  Q  C  R  H 241atttggcttgacagtgtttcttgccatggacacgagtctgctctctggcagtgtagacac 101H  E  W  G  K  H  Y  C  N  H  N  E  D  A  G  V  T  C  S  D 301catgaatggggaaagcattattgcaatcataatgaagatgctggtgtgacatgttctgat 121G  S  D  L  E  L  R  L  K  G  G  G  S  H  C  A  G  T  V  E 361ggatcagatctggaactgagacttaaaggtggaggcagccactgtgctgggacagtggag 141V  E  I  Q  K  L  V  G  K  V  C  D  R  S  W  G  L  K  E  A 421gtggaaattcagaaactggtaggaaaagtgtgtgatagaagctggggactgaaagaagct 161D  V  V  C  R  Q  L  G  C  G  S  A  L  K  T  S  Y  Q  V  Y 481gatgtggtttgcaggcagctgggatgtggatctgcactcaaaacatcatatcaagtttat 181S  K  T  K  A  T  N  T  W  L  F  V  S  S  C  N  G  N  E  T 541tccaaaaccaaggcaacaaacacatggctgtttgtaagcagctgtaatggaaatgaaact 201S  L  W  D  C  K  N  W  Q  W  G  G  L  S  C  D  H  Y  D  E 601tctctttgggactgcaagaattggcagtggggtggacttagttgtgatcactatgacgaa 221A  K  I  T  C  S  A  H  R  K  P  R  L  V  G  G  D  I  P  C 661gccaaaattacctgctcagcccacaggaaacccaggctggttggaggggacattccctgc 241S  G  R  V  E  V  Q  H  G  D  T  W  G  T  V  C  D  S  D  F 721tctggtcgtgttgaagtacaacatggagacacgtggggcaccgtctgtgattctgacttc 261S  L  E  A  A  S  V  L  C  R  E  L  Q  C  G  T  V  V  S  L 781tctctggaggcggccagcgtgctgtgcagggaactacagtgcggcactgtggtttccctc 281L  G  G  A  H  F  G  E  G  S  G  Q  I  W  A  E  E  F  Q  C 841ctggggggagctcactttggagaaggaagtggacagatctgggctgaagaattccagtgt 301E  G  H  E  S  H  L  S  L  C  P  V  A  P  R  P  D  G  T  C 901gaggggcacgagtcccacctttcactctgcccagtagcaccccgccctgacgggacatgt 321S  H  S  R  D  V  G  V  V  C  S  R  Y  T  Q  I  R  L  V  N 961agccacagcagggacgtcggcgtagtctgctcaagatacacacaaatccgcttggtgaat 341G  K  T  P  C  E  G  R  V  E  L  N  I  L  G  S  W  G  S  L 1021ggcaagaccccatgtgaaggaagagtggagctcaacattcttgggtcctgggggtccctc 361C  N  S  H  W  D  M  E  D  A  H  V  L  C  Q  Q  L  K  C  G 1081tgcaactctcactgggacatggaagatgcccatgttttatgccagcagcttaaatgtgga 381V  A  L  S  I  P  G  G  A  P  F  G  K  G  S  E  Q  V  W  R 1141gttgccctttctatcccgggaggagcaccttttgggaaaggaagtgagcaggtctggagg 401H  M  F  H  C  T  G  T  E  K  H  M  G  D  C  S  V  T  A  L 1201cacatgtttcactgcactgggactgagaagcacatgggagattgttccgtcactgctctg 421G  A  S  L  C  S  S  G  Q  V  A  S  V  I  C  S  G  N  Q  S 1261ggcgcatcactctgttcttcagggcaagtggcctctgtaatctgctcagggaaccagagt 441Q  T  L  S  P  C  N  S  S  S  S  D  P  S  S  S  I  I  S  E 1321cagacactatccccgtgcaattcatcatcctcggacccatcaagctctattatttcagaa 461E  N  G  V  A  C  I  G  S  G  Q  L  R  L  V  D  G  G  G  R 1381gaaaatggtgttgcctgcatagggagtggtcaacttcgcctggtcgatggaggtggtcgt 481C  A  G  R  V  E  V  Y  H  E  G  S  W  G  T  I  C  D  D  S 1441tgtgctgggagagtagaggtctatcatgagggctcctggggcaccatctgtgatgacagc 501W  D  L  N  D  A  H  V  V  C  K  Q  L  S  C  G  W  A  I  N 1501tgggacctgaatgatgcccatgtggtgtgcaaacagctgagctgtggatgggccattaat 521A  T  G  S  A  H  F  G  E  G  T  G  P  I  W  L  D  E  I  N 1561gccactggttctgctcattttggggaaggaacagggcccatttggctggatgagataaac 541C  N  G  K  E  S  H  I  W  Q  C  H  S  H  G  W  G  R  H  N 1621tgtaatggaaaagaatctcatatttggcaatgccactcacatggttgggggcggcacaat 561C  R  H  K  E  D  A  G  V  I  C  S  E  F  M  S  L  R  L  I 1681tgcaggcataaggaggatgcaggagtcatctgctcggagttcatgtctctcagactgatc 581S  E  N  S  R  E  T  C  A  G  R  L  E  V  F  Y  N  G  A  W 1741agtgaaaacagcagagagacctgtgcagggcgcctggaagttttttacaacggagcttgg 601G  S  V  G  K  N  S  M  S  P  A  T  V  G  V  V  C  R  Q  L 1801ggcagcgttggcaagaatagcatgtctccagccacagtgggggtggtatgcaggcagctg 621G  C  A  D  R  G  D  I  S  P  A  S  S  D  K  T  V  S  R  H 1861ggctgtgcagacagaggggacatcagccctgcatcttcagacaagacagtgtccaggcac 641M  W  V  D  N  V  Q  C  P  K  G  P  D  T  L  W  Q  C  P  S 1921atgtgggtggacaatgttcagtgtcctaaaggacctgacaccctatggcagtgcccatca 661S  P  W  K  K  R  L  A  S  P  S  E  E  T  W  I  T  C  A  N 1981tctccatggaagaagagactggccagcccctcagaggagacatggatcacatgtgccaac 681K  I  R  L  Q  E  G  N  T  N  C  S  G  R  V  E  I  W  Y  G 2041aaaataagacttcaagaaggaaacactaattgttctggacgtgtggagatctggtacgga 701G  S  W  G  T  V  C  D  D  S  W  D  L  E  D  A  Q  V  V  C 2101ggttcctggggcactgtgtgtgacgactcctgggaccttgaagatgctcaggtggtgtgc 721R  Q  L  G  C  G  S  A  L  E  A  G  K  E  A  A  F  G  Q  G 2161cgacagctgggctgtggctcagctttggaggcaggaaaagaggccgcatttggccagggg 741T  G  P  I  W  L  N  E  V  K  C  K  G  N  E  T  S  L  W  D 2221actgggcccatatggctcaatgaagtgaagtgcaaggggaatgaaacctccttgtgggat 761C  P  A  R  S  W  G  H  S  D  C  G  H  K  E  D  A  A  V  T 2281tgtcctgccagatcctggggccacagtgactgtggacacaaggaggatgctgctgtgacg 781C  S  E  I  A  K  S  R  E  S  L  H  A  T  G  R  S  S  F  V 2341tgttcagaaattgcaaagagccgagaatccctacatgccacaggtcgctcatcttttgtt 801A  L  A  I  F  G  V  I  L  L  A  C  L  I  A  F  L  I  W  T 2401gcacttgcaatctttggggtcattctgttggcctgtctcatcgcattcctcatttggact 821Q  K  R  R  Q  R  Q  R  L  S  V  F  S  G  G  E  N  S  V  H 2461cagaagcgaagacagaggcagcggctctcagttttctcaggaggagagaattctgtccat 841Q  I  Q  Y  R  E  M  N  S  C  L  K  A  D  E  T  D  M  L  N 2521caaattcaataccgggagatgaattcttgcctgaaagcagatgaaacggatatgctaaat 861P  S  G  D  H  S  E  V  Q 2581 ccctcaggagaccactctgaagtacaa 1 MDKLRMVLHENSGSADLKLR VVDGVTECSG RLEVKFQGEW GTICDDGWDS SEQ ID 51 DDAAVACKQLGCPTAVTAIG RVNASEGTGH IWLDSVSCHG HESALWQCRH NO: 2 101 HEWGKHYCNHNEDAGVTCSD GSDLELRLKG GGSHCAGTVE VEIQKLVGKV 151 CDRSWGLKEA DVVCRQLGCGSALKTSYQVY SKTKATNTWL FVSSCNGNET 201 SLWDCKNWQW GGLSCDHYDE AKITCSAHRKPRLVGGDIPC SGRVEVQHGD 251 TWGTVCDSDF SLEAASVLCR ELQCGTVVSL LGGAHFGEGSGQIWAEEFQC 301 EGHESHLSLC PVAPRPDGTC SHSRDVGVVC SRYTQIRLVN GKTPCEGRVE351 LNILGSWGSL CNSHWDMEDA HVLCQQLKCG VALSIPGGAP FGKGSEQVWR 401HMFHCTGTEK HMGDCSVTAL GASLCSSGQV ASVICSGNQS QTLSPCNSSS 451 SDPSSSIISEENGVACIGSG QLRLVDGGGR CAGRVEVYHE GSWGTICDDS 501 WDLNDAHVVC KQLSCGWAINATGSAHFGEG TGPIWLDEIN CNGKESHIWQ 551 CHSHGWGRHN CRHKEDAGVI CSEFMSLRLISENSRETCAG RLEVFYNGAW 601 GSVGKNSMSP ATVGVVCRQL GCADRGDISP ASSDKTVSRHMWVDNVQCPK 651 GPDTLWQCPS SPWKKRLASP SEETWITCAN KIRLQEGNTN CSGRVEIWYG701 GSWGTVCDDS WDLEDAQVVC RQLGCGSALE AGKEAAFGQG TGPIWLNEVK 751CKGNETSLWD CPARSWGHSD CGHKEDAAVT CSEIAKSRES LHATGRSSFV 801 ALAIFGVILLACLIAFLIWT QKRRQRQRLS VFSGGENSVH QIQYREMNSC 851 LKADETDMLN PSGDHSEVQ

Example 2 Construction of Plasmid pCMVsusCD163v1

Construction of the plasmid pCMVsusCD163v1 was performed as follows. Thefunctional clone identified in the primary porcine macrophage cDNAlibrary as conferring PRRSV permissivity served as template for PCRamplification of the CD163 insert, including the 5′ and 3′ untranslatedregions, using the primers 5′DS-CD163 (SEQ ID NO:6)(5′-CGGAATTCCGCGGATGTAATAATACAAGAAGA-3′) and 3′CD163 (SEQ ID NO:7)(5′CCGCTCGAGTAGTCCAGGTCTTCATCAAGGTATCTT-3′). Primer 5′DS-CD163incorporates a SacII restriction site at the 5′ end of the CD163 insert,while primer 3′CD163 incorporates an XhoI restriction site at the 3′ endof the insert (underlined). Reactions containing 190 ng of plasmidtemplate were amplified using Platinum pfx DNA polymerase (Invitrogencat #11708-013) following the manufacture's instructions. Reactions wereheated to 94° for 2 minutes then cycled 35 times through 94° for 20seconds, 55° for 30 seconds, and 68° for 3.5 minutes followed by aterminal extension at 72° for 7 minutes. The resulting PCR products werepurified using the Qiaquick PCR purification kit (Qiagen cat #28104),digested with restriction enzymes SacII and XhoI, and the resultingfragments were gel purified using the Qiaquick Gel Extraction kit(Qiagen cat #28704). The CD163 PCR fragment was then ligated into theplasmid pCMV-Script (Stratagene cat #212220) prepared to accept thedigested PCR fragment by digestion with SacII and XhoI followed by gelpurification as described above. The ligated material was transformedinto E. coli strain DH5α and recombinants were selected by growth in 50μg/ml kanamycin and identified by restriction analysis. The resultingplasmid, “pCMVsusCD163v1”, contains the internally deleted porcine CD163insert described in Example 1 under the transcriptional control of theeukaryotic CMV promoter and the neomycin/kanamycin resistance gene underthe control of both eukaryotic and prokaryotic promoters.

Example 3 Construction of the pRSV-Script Expression Vector andpRSVsusCD163v1

The plasmid pRc/RSV (Invitrogen) was used as a template for PCRamplification of the RSV promoter. RSV promoter sequence was containedwithin nucleotides 209 through 604 of pRc/RSV. Forward primer PCIRSVLTR(SEQ ID NO:8) (5′-ACACTCGACATGTCGATGTACGGGCCAGATATACGCGT-3′) and reverseprimer VSRRTLSAC (SEQ ID NO: 9)(5′TTCCTTACAGAGCTCGAGGTGCACACCAATGTGGTGAA-3′) were synthesized.Restriction endonuclease Pci I and Sac I recognition sites (underlined)were incorporated into the 5′ and 3′ primers, respectively, for futurecloning. PCR was performed using the HotMaster Taq DNA Polymerase kit(Eppendorf) following the manufacturer's instructions. The reactionscontained 0.9 ng of pRc/RSV plasmid template and 0.3 μM of each primerdescribed above. The reactions were heated to 94° for 2 minutes thencycled 30 times through 94° for 20 seconds, 52° for 10 seconds, and 65°for 1 minute. The resulting PCR fragment was digested with restrictionenzymes Pci I and Sac I, gel purified, and cloned into the plasmidpCMV-Script (Stratagene) that had been similarly digested to remove theCMV promoter sequence. The final construct placed the RSV promoterimmediately upstream of the multiple cloning site, and was named“pRSV-Script”.

The susCD163v1 insert was cloned behind the RSV promoter as follows. ThesusCD163v1 sequence was excised from plasmid pCMVsusCD163v1 byrestriction digestion (Kpn I and Sac II) and gel purified. This fragmentwas ligated into pRSV-Script which had also been digested with the sameenzymes and gel purified. The ligation mixture was transformed into DH5αE. coli and transformants selected using kanamycin at 50 μg/ml. Theclone contained the correct insert was designed “pRSVsusCD163v1”.

Example 4 Cloning and Characterization of a Longer Variant of PorcineCD163 cDNA

Based on the porcine CD163v1 sequence, a forward primer 5′CD163NotIlong(SEQ ID NO:10) (5′CGGTCCGGAGCGGCCGCGATGTAATAATACAAGAAGATTTAAATGG-3′) anda reverse primer 3′CD163 KpnI (SEQ ID NO:11)(5′CGGTTGGTACCCAGCAATATTCTTTTTTATTTAATGCC-3′) were designed using theLasergene PrimerSelect program (DNASTAR Inc., Madison Wis.) foramplification of a full-length porcine CD163 gene. Restrictionendonuclease sites for Not I and Kpn I (underlined) were included in 5′and 3′ primers, respectively, to allow for convenient cloning. Totalcellular RNA was prepared from primary alveolar macrophages (PAM)harvested from lung lavages of healthy pigs. RNA preparation was doneusing the RNeasy mini kit (Qiagen, Valencia, Calif.). RT-PCR reactionswere prepared using the SuperScript one-step RT-PCR for Long Templateskit (Invitrogen, Carlsbad, Calif.) and RT-PCR parameters were set asfollows: 50° C. for 30 min, 94° C. for 2 min, (94° C. 30 sec, 55° C. 30sec and 68° C. 4 min) for 35 cycles, 72° C. for 10 min. PCR productswere analyzed on 0.8% SeaKem GTG agarose gels. RT-PCR products ofvarious sizes were cut from agarose gels and DNA was extracted using theGeneClean kit (QBiogene). These RT-PCR products were cloned into thepCR2.1-TOPO cloning vector (Invitrogen). Clones were analyzed byrestriction enzyme digestion for the presence of an insert. Coloniescontaining inserts were sequenced using Big Dye Terminator Version 1.0Sequence Reaction kit (Applied Biosystems, Foster City, Calif.) andApplied Biosystems 3730 DNA Analyzer (Applied Biosystems) to confirmsequence authenticity. Sequences were edited and assembled using theLasergene EditSeq and SeqMan programs (DNASTAR Inc., Madison Wis.). Oneplasmid with a large insert was designated “pCRsusCD163v2” (pCR2.1containing porcine CD163 variant 2 which we have designated SEQ IDNO:12). The coding sequence contained within SEQ ID NO:12 is reproducedbelow and is designated SEQ ID NO:13. Sequence analysis showed that thisporcine CD163 encodes an amino acid sequence of 1115 amino acids whichwe have designated SEQ ID NO:14. When compared to the porcine CD163sequence in GenBank (Accession No. AJ311716), our CD163v2 sequence is98.9% identical at the amino acid level. CD163v2 also has an additional5 amino acid residues at the extreme 5′ end, extending the open readingframe to an in-frame upstream ATG initiation codon (as in the porcineCD163v1 sequence described in example 1). Porcine CD163 is 84.3%identical to human CD163 (GenBank Accession No. Z22968), and 73.7%identical to mouse CD163 (GenBank Accession No. AF274883) at the aminoacid level. The predicted signal sequence and transmembrane region ofSEQ ID NO:14 are indicated by underlining and bolding respectively. Todetermine whether other CD163 sequences contain similar sequencefeatures is easily determined by inspection of the sequence.

SEQUENCE ID NO gtaataatac aagaagattt aaatggcata aaaccttgga atggacaaactcagaatggt 60 SEQ ID gctacatgaa aactctggat ctgcagactt tagaagatgttctgcccatt taagttcctt 120 NO: 12 cacttttgct gtagtcgctg ttctcagtgcctgcttggtc actagttctc ttggaggaaa 180 agacaaggag ctgaggctaa cgggtggtgaaaacaagtgc tctggaagag tggaggtgaa 240 agtgcaggag gagtggggaa ctgtgtgtaataatggctgg gacatggatg tggtctctgt 300 tgtttgtagg cagctgggat gtccaactgctatcaaagcc actggatggg ctaattttag 360 tgcaggttct ggacgcattt ggatggatcatgtttcttgt cgagggaatg agtcagctct 420 ctgggactgc aaacatgatg gatggggaaagcataactgt actcaccaac aggatgctgg 480 agtaacctgc tcagatggat ctgatttagagatggggctg gtgaatggag gaaaccggtg 540 cttaggaaga atagaagtca aatttcaaggacggtgggga acagtgtgtg atgataactt 600 caacataaat catgcttctg tggtttgtaaacaacttgaa tgtggaagtg ctgtcagttt 660 ctctggttca gctaattttg gagaaggttctggaccaatc tggtttgatg atcttgtatg 720 caatggaaat gagtcagctc tctggaactgcaaacatgaa ggatggggaa agcacaattg 780 cgatcatgct gaggatgctg gagtgatttgcttaaatgga gcagacctga aactgagagt 840 ggtagatgga gtcactgaat gttcaggaagattggaagtg aaattccaag gagaatgggg 900 aacaatctgt gatgatggct gggatagtgatgatgccgct gtggcatgta agcaactggg 960 atgtccaact gctgtcactg ccattggtcgagttaacgcc agtgagggaa ctggacacat 1020 ttggcttgac agtgtttctt gccatggacacgagtctgct ctctggcagt gtagacacca 1080 tgaatgggga aagcattatt gcaatcatgatgaagatgct ggtgtgacat gttctgatgg 1140 atcagatctg gaactgagac ttaaaggtggaggcagccac tgtgctggga cagtggaggt 1200 ggaaattcag aaactggtag gaaaagtgtgtgatagaagc tggggactga aagaagctga 1260 tgtggtttgc aggcagctgg gatgtggatctgcactcaaa acatcatatc aagtttattc 1320 caaaaccaag gcaacaaaca catggctgtttgtaagcagc tgtaatggaa atgaaacttc 1380 tctttgggac tgcaagaatt ggcagtggggtggacttagt tgtgatcact atgacgaagc 1440 caaaattacc tgctcagccc acaggaaacccaggctggtt ggaggggaca ttccctgctc 1500 tggtcgtgtt gaagtacaac atggagacacgtggggcacc gtctgtgatt ctgacttctc 1560 tctggaggcg gccagcgtgc tgtgcagggaactacagtgc ggcactgtgg tttccctcct 1620 ggggggagct cactttggag aaggaagtggacagatctgg gctgaagaat tccagtgtga 1680 ggggcacgag tcccaccttt cactctgcccagtagcaccc cgccctgacg ggacatgtag 1740 ccacagcagg gacgtcggcg tagtctgctcaagatacaca caaatccgct tggtgaatgg 1800 caagacccca tgtgaaggaa gagtggagctcaacattctt gggtcctggg ggtccctctg 1860 caactctcac tgggacatgg aagatgcccatgttttatgc cagcagctta aatgtggagt 1920 tgccctttct atcccgggag gagcaccttttgggaaagga agtgagcagg tctggaggca 1980 catgtttcac tgcactggga ctgagaagcacatgggagat tgttccgtca ctgctctggg 2040 cgcatcactc tgttcttcag ggcaagtggcctctgtaatc tgctcaggga accagagtca 2100 gacactatct ccgtgcaatt catcatcctcggacccatca agctctatta tttcagaaga 2160 aaatggtgtt gcctgcatag ggagtggtcaacttcgcctg gtcgatggag gtggtcgttg 2220 tgctgggaga gtagaggtct atcatgagggctcctggggc accatctgtg atgacagctg 2280 ggacctgaat gatgcccatg tggtgtgcaaacagctgagc tgtggatggg ccattaatgc 2340 cactggttct gctcattttg gggaaggaacagggcccatt tggctggatg agataaactg 2400 taatggaaaa gaatctcata tttggcaatgccactcacat ggttgggggc ggcacaattg 2460 caggcataag gaggatgcag gagtcatctgctcagagttc atgtctctga gactgatcag 2520 tgaaaacagc agagagacct gtgcagggcgcctggaagtt ttttacaacg gagcttgggg 2580 cagcgttggc aggaatagca tgtctccagccacagtgggg gtggtatgca ggcagctggg 2640 ctgtgcagac agaggggaca tcagccctgcatcttcagac aagacagtgt ccaggcacat 2700 gtgggtggac aatgttcagt gtcctaaaggacctgacaca ctatggcagt gcccatcatc 2760 tccatggaag aagagactgg ccagcccctcagaggagaca tggatcacat gtgccaacaa 2820 aataagactt caagaaggaa acactaattgttctggacgt gtggagatct ggtacggagg 2880 ttcctggggc actgtgtgtg acgactcctgggaccttgaa gatgctcagg tggtgtgccg 2940 acagctgggc tgtggctcag ctttggaggcaggaaaagag gccgcatttg gccaggggac 3000 tgggcccata tggctcaatg aagtgaagtgcaaggggaat gaaacctcct tgtgggattg 3060 tcctgccaga tcctggggcc acagtgactgtggacacaag gaggatgctg ctgtgacgtg 3120 ctcagaaatt gcaaagagcc gagaatccctacatgccaca ggtcgctcat cttttgttgc 3180 acttgcaatc tttggggtca ttctgttggcctgtctcatc gcattcctca tttggactca 3240 gaagcgaaga cagaggcagc ggctctcagttttctcagga ggagagaatt ctgtccatca 3300 aattcaatac cgggagatga attcttgcctgaaagcagat gaaacggata tgctaaatcc 3360 ctcaggagac cactctgaag tacaatgaaaaggaaaatgg gaattataac ctggtgagtt 3420 cagcctttaa gataccttga tgaagacctggactattgaa tgagcaagaa tctgcctctt 3480 acactgaaga ttacaataca gtcctctgtctcctggtatt ccaaagactg ctgctgaatt 3540 tctaaagaat agattggtga atgtgactactcaaagttgt atgtaagact ttcaagggca 3600 ttaaataaaa aagaatattg ctg 3623 1M  D  K  L  R  M  V  L  H  E  N  S  G  S  A  D  F  R  R  C SEQ ID 1atggacaaactcagaatggtgctacatgaaaactctggatctgcagactttagaagatgt NO: 13 21S  A  H  L  S  S  F  T  F  A  V  V  A  V  L  S  A  C  L  V and 14 61tctgcccatttaagttccttcacttttgctgtagtcgctgttctcagtgcctgcttggtc 41T  S  S  L  G  G  K  D  K  E  L  R  L  T  G  G  E  N  K  C 121actagttctcttggaggaaaagacaaggagctgaggctaacgggtggtgaaaacaagtgc 61S  G  R  V  E  V  K  V  Q  E  E  W  G  T  V  C  N  N  G  W 181tctggaagagtggaggtgaaagtgcaggaggagtggggaactgtgtgtaataatggctgg 81D  M  D  V  V  S  V  V  C  R  Q  L  G  C  P  T  A  I  K  A 241gacatggatgtggtctctgttgtttgtaggcagctgggatgtccaactgctatcaaagcc 101T  G  W  A  N  F  S  A  G  S  G  R  I  W  M  D  H  V  S  C 301actggatgggctaattttagtgcaggttctggacgcatttggatggatcatgtttcttgt 121R  G  N  E  S  A  L  W  D  C  K  H  D  G  W  G  K  H  N  C 361cgagggaatgagtcagctctctgggactgcaaacatgatggatggggaaagcataactgt 141T  H  Q  Q  D  A  G  V  T  C  S  D  G  S  D  L  E  M  G  L 421actcaccaacaggatgctggagtaacctgctcagatggatctgatttagagatggggctg 161V  N  G  G  N  R  C  L  G  R  I  E  V  K  F  Q  G  R  W  G 481gtgaatggaggaaaccggtgcttaggaagaatagaagtcaaatttcaaggacggtgggga 181T  V  C  D  D  N  F  N  I  N  H  A  S  V  V  C  K  Q  L  E 541acagtgtgtgatgataacttcaacataaatcatgcttctgtggtttgtaaacaacttgaa 201C  G  S  A  V  S  F  S  G  S  A  N  F  G  E  G  S  G  P  I 601tgtggaagtgctgtcagtttctctggttcagctaattttggagaaggttctggaccaatc 221W  F  D  D  L  V  C  N  G  N  E  S  A  L  W  E  C  K  H  E 661tggtttgatgatcttgtatgcaatggaaatgagtcagctctctggaactgcaaacatgaa 241G  W  G  K  H  N  C  D  H  A  E  D  A  G  V  I  C  L  N  G 721ggatggggaaagcacaattgcgatcatgctgaggatgctggagtgatttgcttaaatgga 261A  D  L  K  L  R  V  V  D  G  V  T  E  C  S  G  R  L  E  V 781gcagacctgaaactgagagtggtagatggagtcactgaatgttcaggaagattggaagtg 281K  F  Q  G  E  W  G  T  I  C  D  D  G  W  D  S  D  D  A  A 841aaattccaaggagaatggggaacaatctgtgatgatggctgggatagtgatgatgccgct 301V  A  C  K  Q  L  G  C  P  T  A  V  T  A  I  G  R  V  N  A 901gtggcatgtaagcaactgggatgtccaactgctgtcactgccattggtcgagttaacgcc 321S  E  G  T  G  H  I  W  L  D  S  V  S  C  H  G  H  E  S  A 961agtgagggaactggacacatttggcttgacagtgtttcttgccatggacacgagtctgct 341L  W  Q  C  R  H  H  E  W  G  K  H  Y  C  N  H  D  E  D  A 1021ctctggcagtgtagacaccatgaatggggaaagcattattgcaatcatgatgaagatgct 361G  V  T  C  S  D  G  S  D  L  E  L  R  L  K  G  G  G  S  H 1081ggtgtgacatgttctgatggatcagatctggaactgagacttaaaggtggaggcagccac 381C  A  G  T  V  E  V  E  I  Q  K  L  V  G  K  V  C  D  R  S 1141tgtgctgggacagtggaggtggaaattcagaaactggtaggaaaagtgtgtgatagaagc 401W  G  L  K  E  A  D  V  V  C  R  Q  L  G  C  G  S  A  L  K 1201tggggactgaaagaagctgatgtggtttgcaggcagctgggatgtggatctgcactcaaa 421T  S  Y  Q  V  Y  S  K  T  K  A  T  N  T  W  L  F  V  S  S 1261acatcatatcaagtttattccaaaaccaaggcaacaaacacatggctgtttgtaagcagc 441C  N  G  N  E  T  S  L  W  D  C  K  N  W  Q  W  G  G  L  S 1321tgtaatggaaatgaaacttctctttgggactgcaagaattggcagtggggtggacttagt 461C  D  H  Y  D  E  A  K  I  T  C  S  A  H  R  K  P  R  L  V 1381tgtgatcactatgacgaagccaaaattacctgctcagcccacaggaaacccaggctggtt 481G  G  D  I  P  C  S  G  R  V  E  V  Q  H  G  D  T  W  G  T 1441ggaggggacattccctgctctggtcgtgttgaagtacaacatggagacacgtggggcacc 501V  C  D  S  D  F  S  L  E  A  A  S  V  L  C  R  E  L  Q  C 1501gtctgtgattctgacttctctctggaggcggccagcgtgctgtgcagggaactacagtgc 521G  T  V  V  S  L  L  G  G  A  H  F  G  E  G  S  G  Q  I  W 1561ggcactgtggtttccctcctggggggagctcactttggagaaggaagtggacagatctgg 541A  E  E  F  Q  C  E  G  H  E  S  H  L  S  L  C  P  V  A  P 1621gctgaagaattccagtgtgaggggcacgagtcccacctttcactctgcccagtagcaccc 561R  P  D  G  T  C  S  H  S  R  D  V  G  V  V  C  S  R  Y  T 1681cgccctgacgggacatgtagccacagcagggacgtcggcgtagtctgctcaagatacaca 581Q  I  R  L  V  N  G  K  T  P  C  E  G  R  V  E  L  N  I  L 1741caaatccgcttggtgaatggcaagaccccatgtgaaggaagagtggagctcaacattctt 601G  S  W  G  S  L  C  N  S  H  W  D  M  E  D  A  H  V  L  C 1801gggtcctgggggtccctctgcaactctcactgggacatggaagatgcccatgttttatgc 621Q  Q  L  K  C  G  V  A  L  S  I  P  G  G  A  P  F  G  K  G 1861cagcagcttaaatgtggagttgccctttctatcccgggaggagcaccttttgggaaagga 641S  E  Q  V  W  R  H  M  F  H  C  T  G  T  E  K  H  M  G  D 1921agtgagcaggtctggaggcacatgtttcactgcactgggactgagaagcacatgggagat 661C  S  V  T  A  L  G  A  S  L  C  S  S  G  Q  V  A  S  V  I 1981tgttccgtcactgctctgggcgcatcactctgttcttcagggcaagtggcctctgtaatc 681C  S  G  N  Q  S  Q  T  L  S  P  C  N  S  S  S  S  D  P  S 2041tgctcagggaaccagagtcagacactatctccgtgcaattcatcatcctcggacccatca 701S  S  I  I  S  E  E  N  G  V  A  C  I  G  S  G  Q  L  R  L 2101agctctattatttcagaagaaaatggtgttgcctgcatagggagtggtcaacttcgcctg 721V  D  G  G  G  R  C  A  G  R  V  E  V  Y  H  E  G  S  W  G 2161gtcgatggaggtggtcgttgtgctgggagagtagaggtctatcatgagggctcctggggc 741T  I  C  D  D  S  W  D  L  N  D  A  H  V  V  C  K  Q  L  S 2221accatctgtgatgacagctgggacctgaatgatgcccatgtggtgtgcaaacagctgagc 761C  G  W  A  I  N  A  T  G  S  A  H  F  G  E  G  T  G  P  I 2281tgtggatgggccattaatgccactggttctgctcattttggggaaggaacagggcccatt 781W  L  D  E  I  N  C  E  G  K  E  S  H  I  W  Q  C  H  S  H 2341tggctggatgagataaactgtaatggaaaagaatctcatatttggcaatgccactcacat 801G  W  G  R  H  N  C  R  H  K  E  D  A  G  V  I  C  S  E  F 2401ggttgggggcggcacaattgcaggcataaggaggatgcaggagtcatctgctcagagttc 821M  S  L  R  L  I  S  E  N  S  R  E  T  C  A  G  R  L  E  V 2461atgtctctgagactgatcagtgaaaacagcagagagacctgtgcagggcgcctggaagtt 841F  Y  N  G  A  W  G  S  V  G  R  N  S  M  S  P  A  T  V  G 2521ttttacaacggagcttggggcagcgttggcaggaatagcatgtctccagccacagtgggg 861V  V  C  R  Q  L  G  C  A  D  R  G  D  I  S  P  A  S  S  D 2581gtggtatgcaggcagctgggctgtgcagacagaggggacatcagccctgcatcttcagac 881K  T  V  S  R  H  M  W  V  D  N  V  Q  C  P  K  G  P  D  T 2641aagacagtgtccaggcacatgtgggtggacaatgttcagtgtcctaaaggacctgacaca 901L  W  Q  C  P  S  S  P  W  K  K  R  L  A  S  P  S  E  E  T 2701ctatggcagtgcccatcatctccatggaagaagagactggccagcccctcagaggagaca 921W  I  T  C  A  N  K  I  R  L  Q  E  G  N  I  N  C  S  G  R 2761tggatcacatgtgccaacaaaataagacttcaagaaggaaacactaattgttctggacgt 941V  E  I  W  Y  G  G  S  W  G  T  V  C  D  D  S  W  D  L  E 2821gtggagatctggtacggaggttcctggggcactgtgtgtgacgactcctgggaccttgaa 961D  A  Q  V  V  C  R  Q  L  G  C  G  S  A  L  E  A  G  K  E 2881gatgctcaggtggtgtgccgacagctgggctgtggctcagctttggaggcaggaaaagag 981A  A  F  G  Q  G  T  G  P  I  W  L  N  E  V  K  C  K  G  N 2941gccgcatttggccaggggactgggcccatatggctcaatgaagtgaagtgcaaggggaat 1001E  T  S  L  W  D  C  P  A  R  S  W  G  H  S  D  C  G  H  K 3001gaaacctccttgtgggattgtcctgccagatcctggggccacagtgactgtggacacaag 1021E  D  A  A  V  T  C  S  E  I  A  K  S  R  E  S  L  H  A  T 3061gaggatgctgctgtgacgtgctcagaaattgcaaagagccgagaatccctacatgccaca 1041G  R  S  S  F  V  A  L  A  I  F  G  V  I  L  L  A  C  L  I 3121ggtcgctcatcttttgttgcacttgcaatctttggggtcattctgttggcctgtctcatc 1061A  F  L  I  W  T  Q  K  R  R  Q  R  Q  R  L  S  V  F  S  G 3181gcattcctcatttggactcagaagcgaagacagaggcagcggctctcagttttctcagga 1081G  E  N  S  V  H  Q  I  Q  Y  R  E  M  N  S  C  L  K  A  D 3241ggagagaattctgtccatcaaattcaataccgggagatgaattcttgcctgaaagcagat 1101E  T  D  M  L  N  P  S  G  D  H  S  E  V  Q 3301gaaacggatatgctaaatccctcaggagaccactctgaagtacaa 1MDKLRMVLHE NSGSADFRRC SAHLSSFTFA VVAVLSACLV TSSLGGKDKE SEQ ID 51LRLTGGENKC SGRVEVKVQE EWGTVCNNGW DMDVVSVVCR QLGCPTAIKA NO: 14 101TGWANFSAGS GRIWMDHVSC RGNESALWDC KHDGWGKHNC THQQDAGVTC 151 SDGSDLEMGLVNGGNRCLGR IEVKFQGRWG TVCDDNFNIN HASVVCKQLE 201 CGSAVSFSGS ANFGEGSGPIWFDDLVCNGN ESALWNCKHE GWGKHNCDHA 251 EDAGVICLNG ADLKLRVVDG VTECSGRLEVKFQGEWGTIC DDGWDSDDAA 301 VACKQLGCPT AVTAIGRVNA SEGTGHIWLD SVSCHGHESALWQCRHHEWG 351 KHYCNHDEDA GVTCSDGSDL ELRLKGGGSH CAGTVEVEIQ KLVGKVCDRS401 WGLKEADVVC RQLGCGSALK TSYQVYSKTK ATNTWLFVSS CNGNETSLWD 451CKNWQWGGLS CDHYDEAKIT CSAHRKPRLV GGDIPCSGRV EVQHGDTWGT 501 VCDSDFSLEAASVLCRELQC GTVVSLLGGA HFGEGSGQIW AEEFQCEGHE 551 SHLSLCPVAP RPDGTCSHSRDVGVVCSRYT QIRLVNGKTP CEGRVELNIL 601 GSWGSLCNSH WDMEDAHVLC QQLKCGVALSIPGGAPFGKG SEQVWRHMFH 651 CTGTEKHMGD CSVTALGASL CSSGQVASVI CSGNQSQTLSPCNSSSSDPS 701 SSIISEENGV ACIGSGQLRL VDGGGRCAGR VEVYHEGSWG TICDDSWDLN751 DAHVVCKQLS CGWAINATGS AHFGEGTGPI WLDEINCNGK ESHIWQCHSH 801GWGRHNCRHK EDAGVICSEF MSLRLISENS RETCAGRLEV FYNGAWGSVG 851 RNSMSPATVGVVCRQLGCAD RGDISPASSD KTVSRHMWVD NVQCPKGPDT 901 LWQCPSSPWK KRLASPSEETWITCANKIRL QEGNTNCSGR VEIWYGGSWG 951 TVCDDSWDLE DAQVVCRQLG CGSALEAGKEAAFGQGTGPI WLNEVKCKGN 1001 ETSLWDCPAR SWGHSDCGHK EDAAVTCSEI AKSRESLHATCRSSFVALAI 1051 FGVILLACLI AFLIWTQKRR QRQRLSVFSG GENSVHQIQY REMNSCLKAD1101 ETDMLNPSGD HSEVQ

Sus CD163 v2 in pCRsusCD163v2 was liberated from pCR2.1 vector afterrestriction enzymes Kpn I and Not I digestion and gel purification.Recipient vector pCMV-script was also cut with the same restrictionenzyme pair and allowed for directional cloning of susCD163v2 into thepCMV-script. After ligation of susCD163 v2 with pCMV-script, the ligatedmixture was used to transform STBL 2 E. coli cells (Invitrogen). Onetransformant was found to contain the CD163 gene by restriction enzymedigestion analysis and was designed pCMV-script susCD163v2 clone#3.

Example 5 Preparation of a RSV Promoter Based Expression System byDirect Ligation and Transfection Method

A non-cloning based procedure to generate microgram quantities of linearDNA suitable for use in generating stable cell lines expressing CD163from an RSV promoter was developed (FIG. 4). The procedure involves theisolation and ligation of two pieces of DNA, one containing the neomycingene and RSV promoter cassette derived from pRSV-script, and the othercontaining the susCD163v2 coding sequence from pCMVsusCD163v2. Vectorplasmid pRSV-Script was linearized with DraIII upstream of the neomycingene, and bunted with the Klenow fragment of E. coli DNA polymerase.This plasmid was then digested with Non immediately downstream of theRSV promoter. The pCMVsusCD163v2 clone was digested in the vectorsequence downstream of the CD163 insert with DrdI, and blunted withKlenow fragment of DNA polymerase. The CD163 coding sequence wasliberated from the vector with a NotI located immediately upstream ofthe CD163 coding sequence. For each plasmid digestion the appropriatefragments were purified from agarose gels. A large-scale ligationreaction was performed as follows. Approximately 20 μg of each DNAfragment was incubated in a volume of 600 μL with 15 units of T4 DNAligase. The reaction was incubated at room temperature for 20 minutes,at which time an aliquot was removed and the reaction frozen on dry ice.Agarose gel analysis of the aliquot revealed that a significant amountof non-ligated DNA remained, so another 15 units of ligase was added andincubated for another 10 minutes at room temperature. Followingligation, a linear piece of DNA containing all of the appropriateelements was purified by agarose gel electrophoresis. Ligation of thetwo DNA fragments via the cohesive Not I termini resulted in theplacement of the 5′ sequences of the CD163 gene downstream of the RSVpromoter, allowing for directed expression of CD163 in mammalian cells.Once isolated, the purified DNA was used to transfect various mammaliancell lines.

Example 6 Cloning and Characterization of Human CD163 cDNA

Based on a known human CD163 cDNA sequence (GenBank Accession No.BC051281), a forward primer Hu5′Not (SEQ ID NO: 15)(5′CACCGCGGCCGCGAAGTTATAAATCGCCACCATGAGCAAACTCAGAAT GG-3′) and a reverseprimer Hu3′Kpn (SEQ ID NO: 16)(5′-TGCTCCGGTACCTAGTCCAGGTCTTCATCAAGGTATCTTA-3′) were designed using thePrimerSelect program. Restriction sites for NotI and kpnI (underlined)were incorporated into the 5′ and 3′ primers, respectively, tofacilitate cloning into expression vectors. The sequence CACC was addedto the 5′ end of the 5′ primer to allow directional cloning into thepcDNA3.1D/V5/His/TOPO vector (Cat. No. K49001, Invitrogen, see FIG. 6).Human CD163 cDNAs were amplified from RNA extracted from the U937 cellline after stimulated with phorbol 12-myristate 13-acetate (100 ng/ml)for 3 days. Total cellular RNA was prepared using the RNeasy kit(Qiagen). RT-PCR reactions and sequencing methods were the same asdescribed in Example 4. PCR products were separated on 0.8% SeaKemagarose gel and extracted from the gel using the GeneClean kit. PCRproducts were cloned directionally into the pcDNA3.1D/V5/His/TOPO vectorfollowing the manufacturer's instructions. Two clones with large insertswere sequenced. Sequencing and sequence analysis methods were describedin Example 4. A clone with a correct insert was designed“pcDNA3.1D-humCD163v2” and we have designated the sequence of the insertSEQ ID NO: 17

The CD163 open reading frame in pcDNA3.1D-humCD163v2 is 1121 residues inlength (designated SEQ ID NO: 18 which encodes SEQ ID NO:19 disclosedbelow), and is 100% identical to Genbank Z22968 (a human CD163 cDNA ofthe same length). Our human CD163v2 sequence is also 100% identical toGenbank BC051281 and Z22969 (splice variants of human CD163) except that42 nonhomologous residues in the two Genbank sequences replace the sevencarboxy-terminal residues of our sequence. This difference is due to thepresence of an 83-nucleotide exon in BC051281 and Z22969, and theresulting frame shift at the 3′ end of the exon. (Law, S. K., Micklem,K. J., Shaw, J. M., Zhang, X. P., Dong, Y., Willis, A. C. and Mason, D.Y. (1993) A new macrophage differentiation antigen which is a member ofthe scavenger receptor superfamily. European Journal of Immunology 23(9), 2320-2325).

SEQUENCE ID NO atgagcaaac tcagaatggt gctacttgaa gactctggat ctgctgacttcagaagacat 60 SEQ ID tttgtcaacc tgagtccctt caccattact gtggtcttacttctcagtgc ctgttttgtc 120 NO: 17 accagttctc ttggaggaac agacaaggagctgaggctag tggatggtga aaacaagtgt 180 agcgggagag tggaagtgaa agtccaggaggagtggggaa cggtgtgtaa taatggctgg 240 agcatggaag cggtctctgt gatttgtaaccagctgggat gtccaactgc tatcaaagcc 300 cctggatggg ctaattccag tgcaggttctggacgcattt ggatggatca tgtttcttgt 360 cgtgggaatg agtcagctct ttgggattgcaaacatgatg gatggggaaa gcatagtaac 420 tgtactcacc aacaagatgc tggagtgacctgctcagatg gatccaattt ggaaatgagg 480 ctgacgcgtg gagggaatat gtgttctggaagaatagaga tcaaattcca aggacggtgg 540 ggaacagtgt gtgatgataa cttcaacatagatcatgcat ctgtcatttg tagacaactt 600 gaatgtggaa gtgctgtcag tttctctggttcatctaatt ttggagaagg ctctggacca 660 atctggtttg atgatcttat atgcaacggaaatgagtcag ctctctggaa ctgcaaacat 720 caaggatggg gaaagcataa ctgtgaccatgctgaggatg ctggagtgat ttgctcaaag 780 ggagcagatc tgagcctgag actggtagatggagtcactg aatgttcagg aagattagaa 840 gtgagattcc aaggggaatg ggggacaatatgtgatgacg gctgggacag ttacgatgct 900 gctgtggcat gcaagcaact gggatgtccaactgccgtca cagccattgg tcgagttaac 960 gccagtaagg gatttggaca catctggcttgacagcgttt cttgccaggg acatgaacct 1020 gctgtctggc aatgtaaaca ccatgaatggggaaagcatt attgcaatca caatgaagat 1080 gctggcgtga catgttctga tggatcagatctggagctaa gacttagagg tggaggcagc 1140 cgctgtgctg ggacagttga ggtggagattcagagactgt tagggaaggt gtgtgacaga 1200 ggctggggac tgaaagaagc tgatgtggtttgcaggcagc tgggatgtgg atctgcactc 1260 aaaacatctt atcaagtgta ctccaaaatccaggcaacaa acacatggct gtttctaagt 1320 agctgtaacg gaaatgaaac ttctctttgggactgcaaga actggcaatg gggtggactt 1380 acctgtgatc actatgaaga agccaaaattacctgctcag cccacaggga acccagactg 1440 gttggagggg acattccctg ttctggacgtgttgaagtga agcatggtga cacgtggggc 1500 tccatctgtg attcggactt ctctctggaagctgccagcg ttctatgcag ggaattacag 1560 tgtggcacag ttgtctctat cctggggggagctcactttg gagagggaaa tggacagatc 1620 tgggctgaag aattccagtg tgagggacatgagtcccatc tttcactctg cccagtagca 1680 ccccgcccag aaggaacttg tagccacagcagggatgttg gagtagtctg ctcaagatac 1740 acagaaattc gcttggtgaa tggcaagaccccgtgtgagg gcagagtgga gctcaaaacg 1800 cttggtgcct ggggatccct ctgtaactctcactgggaca tagaagatgc ccatgttctt 1860 tgccagcagc ttaaatgtgg agttgccctttctaccccag gaggagcacg ttttggaaaa 1920 ggaaatggtc agatctggag gcatatgtttcactgcactg ggactgagca gcacatggga 1980 gattgtcctg taactgctct aggtgcttcattatgtcctt cagagcaagt ggcctctgta 2040 atctgctcag gaaaccagtc ccaaacactgtcctcgtgca attcatcgtc tttgggccca 2100 acaaggccta ccattccaga agaaagtgctgtggcctgca tagagagtgg tcaacttcgc 2160 ctggtaaatg gaggaggtcg ctgtgctgggagagtagaga tctatcatga gggctcctgg 2220 ggcaccatct gtgatgacag ctgggacctgagtgatgccc acgtggtttg cagacagctg 2280 ggctgtggag aggccattaa tgccactggttctgctcatt ttggggaagg aacagggccc 2340 atctggctgg atgagatgaa atgcaatggaaaagaatccc gcatttggca gtgccattca 2400 cacggctggg ggcagcaaaa ttgcaggcacaaggaggatg cgggagttat ctgctcagaa 2460 ttcatgtctc tgagactgac cagtgaagccagcagagagg cctgtgcagg gcgtctggaa 2520 gttttttaca atggagcttg gggcactgttggcaagagta gcatgtctga aaccactgtg 2580 ggtgtggtgt gcaggcagct gggctgtgcagacaaaggga aaatcaaccc tgcatcttta 2640 gacaaggcca tgtccattcc catgtgggtggacaatgttc agtgtccaaa aggacctgac 2700 acgctgtggc agtgcccatc atctccatgggagaagagac tggccagccc ctcggaggag 2760 acctggatca catgtgacaa caagataagacttcaggaag gacccacttc ctgttctgga 2820 cgtgtggaga tctggcatgg aggttcctgggggacagtgt gtgatgactc ttgggacttg 2880 gacgatgctc aggtggtgtg tcaacaacttggctgtggtc cagctttgaa agcattcaaa 2940 gaagcagagt ttggtcaggg gactggaccgatatggctca atgaagtgaa gtgcaaaggg 3000 aatgagtctt ccttgtggga ttgtcctgccagacgctggg gccatagtga gtgtgggcac 3060 aaggaagacg ctgcagtgaa ttgcacagatatttcagtgc agaaaacccc acaaaaagcc 3120 acaacaggtc gctcatcccg tcagtcatcctttattgcag tcgggatcct tggggttgtt 3180 ctgttggcca ttttcgtcgc attattcttcttgactaaaa agcgaagaca gagacagcgg 3240 cttgcagttt cctcaagagg agagaacttagtccaccaaa ttcaataccg ggagatgaat 3300 tcttgcctga atgcagatga tctggacctaatgaattcct caggaggcca ttctgagcca 3360 cactgaaaag gaaaatggga atttataacccagtgagttc agcctttaag ataccttgat 3420 gaagacctgg acta 3434 1M  S  K  L  R  M  V  L  L  E  D  S  G  S  A  D  F  R  R  H SEQ ID 1atgagcaaactcagaatggtgctacttgaagactctggatctgctgacttcagaagacat NO: 18 21F  V  N  L  S  P  F  T  I  T  V  V  L  L  L  S  A  C  F  V and 19 61tttgtcaacctgagtcccttcaccattactgtggtcttacttctcagtgcctgttttgtc 41T  S  S  L  G  G  T  D  K  E  L  R  L  V  D  G  E  N  K  C 121accagttctcttggaggaacagacaaggagctgaggctagtggatggtgaaaacaagtgt 61S  G  R  V  E  V  K  V  Q  E  E  W  G  T  V  C  N  N  G  W 181agcgggagagtggaagtgaaagtccaggaggagtggggaacggtgtgtaataatggctgg 81S  M  E  A  V  S  V  I  C  N  Q  L  G  C  P  T  A  I  K  A 241agcatggaagcggtctctgtgatttgtaaccagctgggatgtccaactgctatcaaagcc 101P  G  W  A  N  S  S  A  G  S  G  R  I  W  M  D  H  V  S  C 301cctggatgggctaattccagtgcaggttctggacgcatttggatggatcatgtttcttgt 121R  G  N  E  S  A  L  W  D  C  K  H  D  G  W  G  K  H  S  N 361cgtgggaatgagtcagctctttgggattgcaaacatgatggatggggaaagcatagtaac 141C  T  H  Q  Q  D  A  G  V  T  C  S  D  G  S  N  L  E  M  R 421tgtactcaccaacaagatgctggagtgacctgctcagatggatccaatttggaaatgagg 161L  T  R  G  G  N  M  C  S  G  R  I  E  I  K  F  Q  G  R  W 481ctgacgcgtggagggaatatgtgttctggaagaatagagatcaaattccaaggacggtgg 181G  T  V  C  D  D  N  F  N  I  D  H  A  S  V  I  C  R  Q  L 541ggaacagtgtgtgatgataacttcaacatagatcatgcatctgtcatttgtagacaactt 201E  C  G  S  A  V  S  F  S  G  S  S  N  F  G  E  G  S  G  P 601gaatgtggaagtgctgtcagtttctctggttcatctaattttggagaaggctctggacca 221I  W  F  D  D  L  I  C  N  G  N  E  S  A  L  W  N  C  K  H 661atctggtttgatgatcttatatgcaacggaaatgagtcagctctctggaactgcaaacat 241Q  G  W  G  K  H  N  C  D  H  A  E  D  A  G  V  I  C  S  K 721caaggatggggaaagcataactgtgaccatgctgaggatgctggagtgatttgctcaaag 261G  A  D  L  S  L  R  L  V  D  G  V  T  E  C  S  G  R  L  E 781ggagcagatctgagcctgagactggtagatggagtcactgaatgttcaggaagattagaa 281V  R  F  Q  G  E  W  G  T  I  C  D  D  G  W  D  S  Y  D  A 841gtgagattccaaggggaatgggggacaatatgtgatgacggctgggacagttacgatgct 301A  V  A  C  K  Q  L  G  C  P  T  A  V  T  A  I  G  R  V  N 901gctgtggcatgcaagcaactgggatgtccaactgccgtcacagccattggtcgagttaac 321A  S  K  G  F  G  H  I  W  L  D  S  V  S  C  Q  G  H  E  P 961gccagtaagggatttggacacatctggcttgacagcgtttcttgccagggacatgaacct 341A  V  W  Q  C  K  H  H  E  W  G  K  H  Y  C  N  H  N  E  D 1021gctgtctggcaatgtaaacaccatgaatggggaaagcattattgcaatcacaatgaagat 361A  G  V  T  C  S  D  G  S  D  L  E  L  R  L  R  G  G  G  S 1081gctggcgtgacatgttctgatggatcagatctggagctaagacttagaggtggaggcagc 381R  C  A  G  T  V  E  V  E  I  Q  R  L  L  G  K  V  C  D  R 1141cgctgtgctgggacagttgaggtggagattcagagactgttagggaaggtgtgtgacaga 401G  W  G  L  K  E  A  D  V  V  C  R  Q  L  G  C  G  S  A  L 1201ggctggggactgaaagaagctgatgtggtttgcaggcagctgggatgtggatctgcactc 421K  T  S  Y  Q  V  Y  S  K  I  Q  A  T  N  T  W  L  F  L  S 1261aaaacatcttatcaagtgtactccaaaatccaggcaacaaacacatggctgtttctaagt 441S  C  N  G  N  E  T  S  L  W  D  C  K  N  W  Q  W  G  G  L 1321agctgtaacggaaatgaaacttctctttgggactgcaagaactggcaatggggtggactt 461T  C  D  H  Y  E  E  A  K  I  T  C  S  A  H  R  E  P  R  L 1381acctgtgatcactatgaagaagccaaaattacctgctcagcccacagggaacccagactg 481V  G  G  D  I  P  C  S  G  R  V  E  V  K  H  G  D  T  W  G 1441gttggaggggacattccctgttctggacgtgttgaagtgaagcatggtgacacgtggggc 501S  I  C  D  S  D  F  S  L  E  A  A  S  V  L  C  R  E  L  Q 1501tccatctgtgattcggacttctctctggaagctgccagcgttctatgcagggaattacag 521C  G  T  V  V  S  I  L  G  G  A  H  F  G  E  G  N  G  Q  I 1561tgtggcacagttgtctctatcctggggggagctcactttggagagggaaatggacagatc 541W  A  E  E  F  Q  C  E  G  H  E  S  H  L  S  L  C  P  V  A 1621tgggctgaagaattccagtgtgagggacatgagtcccatctttcactctgcccagtagca 561P  R  P  E  G  T  C  S  H  S  R  D  V  G  V  V  C  S  R  Y 1681ccccgcccagaaggaacttgtagccacagcagggatgttggagtagtctgctcaagatac 581T  E  I  R  L  V  N  G  K  T  P  C  E  G  R  V  E  L  K  T 1741acagaaattcgcttggtgaatggcaagaccccgtgtgagggcagagtggagctcaaaacg 601L  G  A  W  G  S  L  C  N  S  H  W  D  I  E  D  A  H  V  L 1801cttggtgcctggggatccctctgtaactctcactgggacatagaagatgcccatgttctt 621C  Q  Q  L  K  C  G  V  A  L  S  T  P  G  G  A  R  F  G  K 1861tgccagcagcttaaatgtggagttgccctttctaccccaggaggagcacgttttggaaaa 641G  N  G  Q  I  W  R  H  M  F  H  C  T  G  T  E  Q  H  M  G 1921ggaaatggtcagatctggaggcatatgtttcactgcactgggactgagcagcacatggga 661D  C  P  V  T  A  L  G  A  S  L  C  P  S  E  Q  V  A  S  V 1981gattgtcctgtaactgctctaggtgcttcattatgtccttcagagcaagtggcctctgta 681I  C  S  G  N  Q  S  Q  T  L  S  S  C  N  S  S  S  L  G  P 2041atctgctcaggaaaccagtcccaaacactgtcctcgtgcaattcatcgtctttgggccca 701T  R  P  T  I  P  E  E  S  A  V  A  C  I  E  S  G  Q  L  R 2101acaaggcctaccattccagaagaaagtgctgtggcctgcatagagagtggtcaacttcgc 721L  V  N  G  G  G  R  C  A  G  R  V  E  I  Y  H  E  G  S  W 2161ctggtaaatggaggaggtcgctgtgctgggagagtagagatctatcatgagggctcctgg 741G  T  I  C  D  D  S  W  D  L  S  D  A  H  V  V  C  R  Q  L 2221ggcaccatctgtgatgacagctgggacctgagtgatgcccacgtggtttgcagacagctg 761G  C  G  E  A  I  N  A  T  G  S  A  H  F  G  E  G  T  G  P 2281ggctgtggagaggccattaatgccactggttctgctcattttggggaaggaacagggccc 781I  W  L  D  E  M  K  C  N  G  K  E  S  R  I  W  Q  C  H  S 2341atctggctggatgagatgaaatgcaatggaaaagaatcccgcatttggcagtgccattca 801H  G  W  G  Q  Q  N  C  R  H  K  E  D  A  G  V  I  C  S  E 2401cacggctgggggcagcaaaattgcaggcacaaggaggatgcgggagttatctgctcagaa 821F  M  S  L  R  L  T  S  E  A  S  R  E  A  C  A  G  R  L  E 2461ttcatgtctctgagactgaccagtgaagccagcagagaggcctgtgcagggcgtctggaa 841V  F  Y  N  G  A  W  G  T  V  G  K  S  S  M  S  E  T  T  V 2521gttttttacaatggagcttggggcactgttggcaagagtagcatgtctgaaaccactgtg 861G  V  V  C  R  Q  L  G  C  A  D  K  G  K  I  N  P  A  S  L 2581ggtgtggtgtgcaggcagctgggctgtgcagacaaagggaaaatcaaccctgcatcttta 881D  K  A  M  S  I  P  M  W  V  D  N  V  Q  C  P  K  G  P  D 2641gacaaggccatgtccattcccatgtgggtggacaatgttcagtgtccaaaaggacctgac 901T  L  W  Q  C  P  S  S  P  W  E  K  R  L  A  S  P  S  E  E 2701acgctgtggcagtgcccatcatctccatgggagaagagactggccagcccctcggaggag 921T  W  I  T  C  D  N  K  I  R  L  Q  E  G  P  T  S  C  S  G 2761acctggatcacatgtgacaacaagataagacttcaggaaggacccacttcctgttctgga 941R  V  E  I  W  H  G  G  S  W  G  T  V  C  D  D  S  W  D  L 2821cgtgtggagatctggcatggaggttcctgggggacagtgtgtgatgactcttgggacttg 961D  D  A  Q  V  V  C  Q  Q  L  G  C  G  P  A  L  K  A  F  K 2881gacgatgctcaggtggtgtgtcaacaacttggctgtggtccagctttgaaagcattcaaa 981E  A  E  F  G  Q  G  T  G  P  I  W  L  N  E  V  K  C  K  G 2941gaagcagagtttggtcaggggactggaccgatatggctcaatgaagtgaagtgcaaaggg 1001N  E  S  S  L  W  D  C  P  A  R  R  W  G  H  S  E  C  G  H 3001aatgagtcttccttgtgggattgtcctgccagacgctggggccatagtgagtgtgggcac 1021K  E  D  A  A  V  N  C  T  D  I  S  V  Q  K  T  P  Q  K  A 3061aaggaagacgctgcagtgaattgcacagatatttcagtgcagaaaaccccacaaaaagcc 1041T  T  G  R  S  S  R  Q  S  S  F  I  A  V  G  I  L  G  V  V 3121acaacaggtcgctcatcccgtcagtcatcctttattgcagtcgggatccttggggttgtt 1061L  L  A  I  F  V  A  L  F  F  L  T  K  K  R  R  Q  R  Q  R 3181ctgttggccattttcgtcgcattattcttcttgactaaaaagcgaagacagagacagcgg 1081L  A  V  S  S  R  G  E  N  L  V  H  Q  I  Q  Y  R  E  M  N 3241cttgcagtttcctcaagaggagagaacttagtccaccaaattcaataccgggagatgaat 1101S  C  L  N  A  D  D  L  D  L  M  N  S  S  G  G  H  S  E  P 3301tcttgcctgaatgcagatgatctggacctaatgaattcctcaggaggccattctgagcca 1121 H 3361cac 1 MSKLRMVLLE DSGSADFRRH FVNLSPFTIT VVLLLSACFV TSSLGGTDKE SEQ ID 51LRLVDGENKC SGRVEVKVQE EWGTVCNNGW SMEAVSVICN QLGCPTAIKA NO: 19 101PGWANSSAGS GRIWMDHVSC RGNESALWDC KHDGWGKHSN CTHQQDAGVT 151 CSDGSNLEMRLTRGGNMCSG RIEIKFQGRW GTVCDDNFNI DHASVICRQL 201 ECGSAVSFSG SSNFGEGSGPIWFDDLICNG NESALWNCKH QGWGKHNCDH 251 AEDAGVICSK GADLSLRLVD GVTECSGRLEVRFQGEWGTI CDDGWDSYDA 301 AVACKQLGCP TAVTAIGRVN ASKGFGHIWL DSVSCQGHEPAVWQCKHHEW 351 GKHYCNHNED AGVTCSDGSD LELRLRGGGS RCAGTVEVEI QRLLGKVCDR401 GWGLKEADVV CRQLGCGSAL KTSYQVYSKI QATNTWLFLS SCNGNETSLW 451DCKNWQWGGL TCDHYEEAKI TCSAHREPRL VGGDIPCSGR VEVKHGDTWG 501 SICDSDFSLEAASVLCRELQ CGTVVSILGG AHFGEGNGQI WAEEFQCEGH 551 ESHLSLCPVA PRPEGTCSHSRDVGVVCSRY TEIRLVNGKT PCEGRVELKT 601 LGAWGSLCNS HWDIEDAHVL CQQLKCGVALSTPGGARFGK GNGQIWRHMF 651 HCTGTEQHMG DCPVTALGAS LCPSEQVASV ICSGNQSQTLSSCNSSSLGP 701 TRPTIPEESA VACIESGQLR LVNGGGRCAG RVEIYHEGSW GTICDDSWDL751 SDAHVVCRQL GCGEAINATG SAHFGEGTGP IWLDEMKCNG KESRIWQCHS 801HGWGQQNCRH KEDAGVICSE FMSLRLTSEA SREACAGRLE VFYNGAWGTV 851 GKSSMSETTVGVVCRQLGCA DKGKINPASL DKAMSIPMWV DNVQCPKGPD 901 TLWQCPSSPW EKRLASPSEETWITCDNKIR LQEGPTSCSG RVEIWHGGSW 951 GTVCDDSWDL DDAQVVCQQL GCGPALKAFKEAEFGQGTGP IWLNEVKCKG 1001 NESSLWDCPA RRWGHSECGH KEDAAVNCTD ISVQKTPQKATTGRSSRQSS 1051 FIAVGILGVV LLAIFVALFF LTKKRRQRQR LAVSSRGENL VHQIQYREMN1101 SCLNADDLDL MNSSGGHSEP H

Example 7 Cloning and Characterization of Murine CD163

Based on the murine CD163 sequence in GenBank (AF274883), a forwardprimer Mus-new5′ (SEQ ID NO: 20)(5′-CACCGCGGCCGCCACACGGAGCCATCAAAATCATCAA-3′) and a reverse primerMus-new3′ (SEQ ID NO:21)(5′-GGTACCGCGAACAAGCAAACCAATAGCAATATTGTTTAATTCCCTC-3′) were designedusing the PrimerSelect program. Restriction endonucleases sites for NotIand KpnI were included in 5′ and 3′ primers, respectively, to allowfuture cloning into other expression vectors. Mouse peritonealmacrophages were harvested from mice 2 days after injectingthioglycollate medium into the peritoneal cavity. Total cellular RNA wasprepared from peritoneal macrophages using the RN easy kit. RT-PCRreactions and RT-PCR parameters were the same as described in Example 4,except the annealing temperature was increased to 60° C. and extensiontemperature increased to 72° C. The PCR product was purified on a 0.8%SeaKem agarose gel and directionally cloned into pcDNA3.1DN5/His/TOPOaccording to the manufacturer's instructions. Several clones with largeinserts were identified for further analysis. A plasmid containing aninsert (SEQ ID NO: 22) with a murine CD163 that encodes a protein of thesame length as (1121 amino acids SEQ ID NO:24) and differs from GenbankAF274883 by only two amino acids (99.8% identity) was designated“pcDNA3.1D-murCD163v2”.

Another plasmid, “pcDNA3.1D-murCD163v3 was generated which contained aninsert (SEQ ID NO: 25) containing a murine CD163 coding sequence (SEQ IDNO: 26) which encodes a protein of 1159 amino acids in length (SEQ IDNO: 27). It differs from AF274883 by only 3 amino acids within the first1107 residues (99.7% identity), but the sequences diverge completelybeginning at residue 1108. This is due to an insertion of 82 nucleotidesin the cDNA, and a concomitant shift in reading frame downstream of theinsertion. As a result, murine CD163v3 contains 52 amino acids at itscarboxy-terminus that are not homologous to the 14 carboxy-terminalresidues of murine CD163v2. These two alternative versions of “fulllength” murine CD163 are most likely splice variant of the same gene, ashas been described for human CD163 (Law, S. K., Micklem, K. J., Shaw, J.M., Zhang, X. P., Dong, Y., Willis, A. C. and Mason, D. Y. (1993) A newmacrophage differentiation antigen which is a member of the scavengerreceptor superfamily. European Journal of Immunology 23 (9), 2320-2325).

SEQUENCE ID NO gctttggaat gggtggacac agaatggttc ttcttggagg tgctggatctcctggttgta 60 SEQ ID aaaggtttgt ccatctaggt ttctttgttg tggctgtgagctcacttctc agtgcctctg 120 NO: 22 ctgtcactaa cgctcctgga gaaatgaagaaggaactgag actggcgggt ggtgaaaaca 180 actgtagtgg gagagtggaa cttaagatccatgacaagtg gggcacagtg tgcagtaacg 240 gctggagcat gaatgaagtg tccgtggtttgccagcagct gggatgccca acttctatta 300 aagcccttgg atgggctaac tccagcgccggctctggata tatctggatg gacaaagttt 360 cttgtacagg gaatgagtca gctctttgggactgcaaaca tgatgggtgg ggaaagcata 420 actgtaccca tgaaaaagat gctggagtgacctgctcaga tggatctaat ttggagatga 480 gactggtgaa cagtgcgggc caccgatgcttaggaagagt agaaataaag ttccagggaa 540 agtgggggac ggtgtgtgac gacaacttcagcaaagatca cgcttctgtg atttgtaaac 600 agcttggatg tggaagtgcc attagtttctctggctcagc taaattggga gctggttctg 660 gaccaatctg gctcgatgac ctggcatgcaatggaaatga gtcagctctc tgggactgca 720 aacaccgggg atggggcaag cataactgtgaccatgctga ggatgtcggt gtgatttgct 780 tagagggagc agatctgagc ctgagactagtggatggagt gtccagatgt tcaggaagat 840 tggaagtgag attccaagga gaatgggggaccgtgtgtga tgataactgg gatctccggg 900 atgcttctgt ggtgtgcaag caactgggatgtccaactgc catcagtgcc attggtcgag 960 ttaatgccag tgagggatct ggacagatttggcttgacaa catttcatgc gaaggacatg 1020 aggcaactct ttgggagtgt aaacaccaagagtggggaaa gcattactgt catcatagag 1080 aagacgctgg cgtgacatgt tctgatggagcagatctgga acttagactt gtaggtggag 1140 gcagtcgctg tgctggcatt gtggaggtggagattcagaa gctgactggg aagatgtgta 1200 gccgaggctg gacactggca gatgcggatgtggtttgcag acagcttgga tgtggatctg 1260 cgcttcaaac ccaggctaag atctactctaaaactggggc aacaaatacg tggctctttc 1320 ctggatcttg taatggaaat gaaactactttttggcaatg caaaaactgg cagtggggcg 1380 gcctttcctg tgataatttc gaagaagccaaagttacctg ctcaggccac agggaaccca 1440 gactggttgg aggagaaatc ccatgctctggtcgtgtgga agtgaaacac ggagacgtgt 1500 ggggctccgt ctgtgatttt gacttgtctctggaagctgc cagtgtggtg tgcagggaat 1560 tacaatgtgg aacagtcgtc tctatcctagggggagcaca ttttggagaa ggaagtggac 1620 agatctgggg tgaagaattc cagtgtagtggggatgagtc ccatctttca ctatgctcag 1680 tggcgccccc gctagacaga acttgtacccacagcaggga tgtcagcgta gtctgctcac 1740 gatacataga tattcgtctg gcaggcggcgagtcctcctg tgagggaaga gtggagctca 1800 agacactcgg agcctggggt cccctctgcagttctcattg ggacatggaa gatgctcatg 1860 tcttatgtca gcagctgaag tgtggggttgcccaatctat tccagaagga gcacattttg 1920 ggaaaggagc tggtcaggtc tggagtcacatgttccactg cactggaact gaggaacata 1980 taggagattg cctcatgact gctctgggtgcgccgacgtg ttccgaagga caggtggcct 2040 ctgtcatctg ctcaggaaac caatcccagacactattgcc atgtagttca ttgtctccag 2100 tccaaacaac aagctctaca attccaaaggagagtgaagt tccctgcata gcaagtggcc 2160 agcttcgctt ggtaggtgga ggtggtcgctgcgctggaag agtggaggtc taccacgagg 2220 gctcttgggg caccgtctgt gatgacaattgggatatgac tgatgccaat gtggtgtgca 2280 agcagctgga ctgtggcgtg gcaattaacgccactggctc tgcttacttc ggggaaggag 2340 caggagctat ctggctagac gaagtcatctgcactgggaa agagtctcat atttggcagt 2400 gccattcaca tggctgggga cgccataactgcaggcacaa agaagatgca ggtgttatct 2460 gctccgagtt catgtctctg aggctgaccaacgaagccca caaagaaaac tgcacaggtc 2520 gccttgaagt gttttacaat ggtacatggggcagtattgg cagtagcaat atgtctccaa 2580 ccactgtggg ggtggtgtgc cgtcagctgggctgtgcaga caacgggact gtgaaaccca 2640 taccttcaga caagacacca tccaggcccatgtgggtaga tcgtgtgcag tgtccaaaag 2700 gagttgacac tttgtggcag tgcccctcgtcaccttggaa acagagacag gccagcccct 2760 cctcccagga gtcctggatc atctgtgacaacaaaataag actccaggaa gggcatacag 2820 actgttctgg acgtgtggag atctggcacaaaggttcctg gggaacagtg tgtgatgact 2880 cctgggatct taatgatgct aaggttgtatgtaagcagtt gggctgtggc caagctgtga 2940 aggcactaaa agaagcagca tttggtccaggaactgggcc catatggctc aatgaaatta 3000 agtgtagagg gaatgagtct tccctgtgggattgtcctgc caaaccgtgg agtcacagcg 3060 actgtgggca caaagaagat gcttccatccagtgcctccc aaaaatgact tcagaatcac 3120 atcatggcac aggtcacccc accctcacggcactcttggt ttgtggagcc attctattgg 3180 tcctcctcat tgtcttcctc ctgtggactctgaagcgacg acagattcag cgacttacag 3240 tttcctcaag aggagaggtc ttgatacatcaagttcagta ccaagagatg gattcaaagg 3300 cggatgatct ggacttgctg aaatcctcgggggtcattca gaggcacact gagaaggaaa 3360 atgataattt ataatccact gaggttggagtttaagaagc cttgacagga cagccagcta 3420 aatggaacaa gagcccaggc aacgcacggatgaccacagc tgcatcttca tgcagtcctt 3480 tgtttcctgg aactctgctg aacctgcaaaaaccatattt gtgaatgtga ccacttaata 3540 gagatgggag actttt 3556 1M  G  G  H  R  M  V  L  L  G  G  A  G  S  P  G  C  K  R  F SEQ ID 1atgggtggacacagaatggttcttcttggaggtgctggatctcctggttgtaaaaggttt NO: 23 21V  H  L  G  F  F  V  V  A  V  S  S  L  L  S  A  S  A  V  T and 61gtccatctaggtttctttgttgtggctgtgagctcacttctcagtgcctctgctgtcact 24 41N  A  P  G  E  M  K  K  E  L  R  L  A  G  G  E  N  N  C  S 121aacgctcctggagaaatgaagaaggaactgagactggcgggtggtgaaaacaactgtagt 61G  R  V  E  L  K  I  H  D  K  W  G  T  V  C  S  N  G  W  S 181gggagagtggaacttaagatccatgacaagtggggcacagtgtgcagtaacggctggagc 81M  N  E  V  S  V  V  C  Q  Q  L  G  C  P  T  S  I  K  A  L 241atgaatgaagtgtccgtggtttgccagcagctgggatgcccaacttctattaaagccctt 101G  W  A  N  S  S  A  G  S  G  Y  I  W  M  D  K  V  S  C  T 301ggatgggctaactccagcgccggctctggatatatctggatggacaaagtttcttgtaca 121G  N  E  S  A  L  W  D  C  K  H  D  G  W  G  K  H  N  C  T 361gggaatgagtcagctctttgggactgcaaacatgatgggtggggaaagcataactgtacc 141H  E  K  D  A  G  V  T  C  S  D  G  S  N  L  E  M  R  L  V 421catgaaaaagatgctggagtgacctgctcagatggatctaatttggagatgagactggtg 161N  S  A  G  H  R  C  L  G  R  V  E  I  K  F  Q  G  K  W  G 481aacagtgcgggccaccgatgcttaggaagagtagaaataaagttccagggaaagtggggg 181T  V  C  D  D  N  F  S  K  D  H  A  S  V  I  C  K  Q  L  G 541acggtgtgtgacgacaacttcagcaaagatcacgcttctgtgatttgtaaacagcttgga 201C  G  S  A  I  S  F  S  G  S  A  K  L  G  A  G  S  G  P  I 601tgtggaagtgccattagtttctctggctcagctaaattgggagctggttctggaccaatc 221W  L  D  D  L  A  C  N  G  N  E  S  A  L  W  D  C  K  H  R 661tggctcgatgacctggcatgcaatggaaatgagtcagctctctgggactgcaaacaccgg 241G  W  G  K  H  N  C  D  H  A  E  D  V  G  V  I  C  L  E  G 721ggatggggcaagcataactgtgaccatgctgaggatgtcggtgtgatttgcttagaggga 261A  D  L  S  L  R  L  V  D  G  V  S  R  C  S  G  R  L  E  V 781gcagatctgagcctgagactagtggatggagtgtccagatgttcaggaagattggaagtg 281R  F  Q  G  E  W  G  T  V  C  D  D  N  W  D  L  R  D  A  S 841agattccaaggagaatgggggaccgtgtgtgatgataactgggatctccgggatgcttct 301V  V  C  K  Q  L  G  C  P  T  A  I  S  A  I  G  R  V  N  A 901gtggtgtgcaagcaactgggatgtccaactgccatcagtgccattggtcgagttaatgcc 321S  E  G  S  G  Q  I  W  L  D  N  I  S  C  E  G  H  E  A  T 961agtgagggatctggacagatttggcttgacaacatttcatgcgaaggacatgaggcaact 341L  W  E  C  K  H  Q  E  W  G  K  H  Y  C  H  H  R  E  D  A 1021ctttgggagtgtaaacaccaagagtggggaaagcattactgtcatcatagagaagacgct 361G  V  T  C  S  D  G  A  D  L  E  L  R  L  V  G  G  G  S  R 1081ggcgtgacatgttctgatggagcagatctggaacttagacttgtaggtggaggcagtcgc 381C  A  G  I  V  E  V  E  I  Q  K  L  T  G  K  M  C  S  R  G 1141tgtgctggcattgtggaggtggagattcagaagctgactgggaagatgtgtagccgaggc 401W  T  L  A  D  A  D  V  V  C  R  Q  L  G  C  G  S  A  L  Q 1201tggacactggcagatgcggatgtggtttgcagacagcttggatgtggatctgcgcttcaa 421T  Q  A  K  I  Y  S  K  T  G  A  T  N  T  W  L  F  P  G  S 1261acccaggctaagatctactctaaaactggggcaacaaatacgtggctctttcctggatct 441C  N  G  N  E  T  T  F  W  Q  C  K  N  W  Q  W  G  G  L  S 1321tgtaatggaaatgaaactactttttggcaatgcaaaaactggcagtggggcggcctttcc 461C  D  N  F  E  E  A  K  V  T  C  S  G  H  R  E  P  R  L  V 1381tgtgataatttcgaagaagccaaagttacctgctcaggccacagggaacccagactggtt 481G  G  E  I  P  C  S  G  R  V  E  V  K  H  G  D  V  W  G  S 1441ggaggagaaatcccatgctctggtcgtgtggaagtgaaacacggagacgtgtggggctcc 501V  C  D  F  D  L  S  L  E  A  A  S  V  V  C  R  E  L  Q  C 1501gtctgtgattttgacttgtctctggaagctgccagtgtggtgtgcagggaattacaatgt 521G  T  V  V  S  I  L  G  G  A  H  F  G  E  G  S  G  Q  I  W 1561ggaacagtcgtctctatcctagggggagcacattttggagaaggaagtggacagatctgg 541G  E  E  F  Q  C  S  G  D  E  S  H  L  S  L  C  S  V  A  P 1621ggtgaagaattccagtgtagtggggatgagtcccatctttcactatgctcagtggcgccc 561P  L  D  R  T  C  T  H  S  R  D  V  S  V  V  C  S  R  Y  I 1681ccgctagacagaacttgtacccacagcagggatgtcagcgtagtctgctcacgatacata 581D  I  R  L  A  G  G  E  S  S  C  E  G  R  V  E  L  K  T  L 1741gatattcgtctggcaggcggcgagtcctcctgtgagggaagagtggagctcaagacactc 601G  A  W  G  P  L  C  S  S  H  W  D  M  E  D  A  H  V  L  C 1801ggagcctggggtcccctctgcagttctcattgggacatggaagatgctcatgtcttatgt 621Q  Q  L  K  C  G  V  A  Q  S  I  P  E  G  A  H  F  G  K  G 1861cagcagctgaagtgtggggttgcccaatctattccagaaggagcacattttgggaaagga 641A  G  Q  V  W  S  H  M  F  H  C  T  G  T  E  E  H  I  G  D 1921gctggtcaggtctggagtcacatgttccactgcactggaactgaggaacatataggagat 661C  L  M  T  A  L  G  A  P  T  C  S  E  G  Q  V  A  S  V  I 1981tgcctcatgactgctctgggtgcgccgacgtgttccgaaggacaggtggcctctgtcatc 681C  S  G  N  Q  S  Q  I  L  L  P  C  S  S  L  S  P  V  Q  T 2041tgctcaggaaaccaatcccagacactattgccatgtagttcattgtctccagtccaaaca 701T  S  S  T  I  P  K  E  S  E  V  P  C  I  A  S  G  Q  L  R 2101acaagctctacaattccaaaggagagtgaagttccctgcatagcaagtggccagcttcgc 721L  V  G  G  G  G  R  C  A  G  R  V  E  V  Y  H  E  G  S  W 2161ttggtaggtggaggtggtcgctgcgctggaagagtggaggtctaccacgagggctcttgg 741G  T  V  C  D  D  N  W  D  M  T  D  A  N  V  V  C  K  Q  L 2221ggcaccgtctgtgatgacaattgggatatgactgatgccaatgtggtgtgcaagcagctg 761D  C  G  V  A  I  N  A  T  G  S  A  Y  F  G  E  G  A  G  A 2281gactgtggcgtggcaattaacgccactggctctgcttacttcggggaaggagcaggagct 781I  W  L  D  E  V  I  C  T  G  K  E  S  H  I  W  Q  C  H  S 2341atctggctagacgaagtcatctgcactgggaaagagtctcatatttggcagtgccattca 801H  G  W  G  R  H  N  C  R  H  K  E  D  A  G  V  I  C  S  E 2401catggctggggacgccataactgcaggcacaaagaagatgcaggtgttatctgctccgag 821F  M  S  L  R  L  I  N  E  A  H  K  E  N  C  T  G  R  L  E 2461ttcatgtctctgaggctgaccaacgaagcccacaaagaaaactgcacaggtcgccttgaa 841V  E  Y  N  G  T  W  G  S  I  G  S  S  N  M  S  P  T  T  V 2521gtgttttacaatggtacatggggcagtattggcagtagcaatatgtctccaaccactgtg 861G  V  V  C  R  Q  L  G  C  A  D  N  G  T  V  K  P  I  P  S 2581ggggtggtgtgccgtcagctgggctgtgcagacaacgggactgtgaaacccataccttca 881D  K  T  P  S  R  P  M  W  V  D  R  V  Q  C  P  K  G  V  D 2641gacaagacaccatccaggcccatgtgggtagatcgtgtgcagtgtccaaaaggagttgac 901T  L  W  Q  C  P  S  S  P  W  K  Q  R  Q  A  S  P  S  S  Q 2701actttgtggcagtgcccctcgtcaccttggaaacagagacaggccagcccctcctcccag 921E  S  W  I  I  C  D  N  K  I  R  L  Q  E  G  H  T  D  C  S 2761gagtcctggatcatctgtgacaacaaaataagactccaggaagggcatacagactgttct 941G  R  V  E  I  W  H  K  G  S  W  G  T  V  C  D  D  S  W  D 2821ggacgtgtggagatctggcacaaaggttcctggggaacagtgtgtgatgactcctgggat 961L  N  D  A  K  V  V  C  K  Q  L  G  C  G  Q  A  V  K  A  L 2881cttaatgatgctaaggttgtatgtaagcagttgggctgtggccaagctgtgaaggcacta 981K  E  A  A  F  G  P  G  T  G  P  I  W  L  N  E  I  K  C  R 2941aaagaagcagcatttggtccaggaactgggcccatatggctcaatgaaattaagtgtaga 1001G  N  E  S  S  L  W  D  C  P  A  K  P  W  S  H  S  D  C  G 3001gggaatgagtcitccctgtgggattgtcctgccaaaccgtggagtcacagcgactgtggg 1021H  K  E  D  A  S  I  Q  C  L  P  K  M  T  S  E  S  H  H  G 3061cacaaagaagatgcttccatccagtgcctcccaaaaatgacttcagaatcacatcatggc 1041T  G  H  P  T  L  T  A  L  L  V  C  G  A  I  L  L  V  L  L 3121acaggtcaccccaccctcacggcactcttggtttgtggagccattctattggtcctcctc 1061I  V  F  L  L  W  T  L  K  R  R  Q  I  Q  R  L  T  V  S  S 3181attgtcttcctcctgtggactctgaagcgacgacagattcagcgacttacagtttcctca 1081R  G  E  V  L  I  H  Q  V  Q  Y  Q  E  M  D  S  K  A  D  D 3241agaggagaggtcttgatacatcaagttcagtaccaagagatggattcaaaggcggatgat 1101L  D  L  L  K  S  S  G  V  I  Q  R  H  T  E  K  E  N  D  N 3301ctggacttgctgaaatcctcgggggtcattcagaggcacactgagaaggaaaatgataat 1121 L 3361tta 1 MGGHRMVLLG GAGSPGCKRF VHLGFFVVAV SSLLSASAVT NAPGEMKKEL SEQ ID 51RLAGGENNCS GRVELKIHDK WGTVCSNGWS MNEVSVVCQQ LGCPTSIKAL NO: 24 101GWANSSAGSG YIWMDKVSCT GNESALWDCK HDGWGKHNCT HEKDAGVTCS 151 DGSNLEMRLVNSAGHRCLGR VEIKFQGKWG TVCDDNFSKD HASVICKQLG 201 CGSAISFSGS AKLGAGSGPIWLDDLACNGN ESALWDCKHR GWGKHNCDHA 251 EDVGVICLEG ADLSLRLVDG VSRCSGRLEVRFQGEWGTVC DDNWDLRDAS 301 VVCKQLGCPT AISAIGRVNA SEGSGQIWLD NISCEGHEATLWECKHQEWG 351 KHYCHHREDA GVTCSDGADL ELRLVGGGSR CAGIVEVEIQ KLTGKMCSRG401 WTLADADVVC RQLGCGSALQ TQAKIYSKTG ATNTWLFPGS CNGNETTFWQ 451CKNWQWGGLS CDNFEEAKVT CSGHREPRLV GGEIPCSGRV EVKHGDVWGS 501 VCDFDLSLEAASVVCRELQC GTVVSILGGA HFGEGSGQIW GEEFQCSGDE 551 SHLSLCSVAP PLDRTCTHSRDVSVVCSRYI DIRLAGGESS CEGRVELKTL 601 GAWGPLCSSH WDMEDAHVLC QQLKCGVAQSIPEGAHFGKG AGQVWSHMFH 651 CTGTEEHIGD CLMTALGAPT CSEGQVASVI CSGNQSQTLLPCSSLSPVQT 701 TSSTIPKESE VPCIASGQLR LVGGGGRCAG RVEVYHEGSW GTVCDDNWDM751 TDANVVCKQL DCGVAINATG SAYFGEGAGA IWLDEVICTG KESHIWQCHS 801HGWGRHNCRH KEDAGVICSE FMSLRLTNEA HKENCTGRLE VFYNGTWGSI 851 GSSNMSPTTVGVVCRQLGCA DNGTVKPIPS DKTPSRPMWV DRVQCPKGVD 901 TLWQCPSSPW KQRQASPSSQESWIICDNKI RLQEGHTDCS GRVEIWHKGS 951 WGTVCDDSWD LNDAKVVCKQ LGCGQAVKALKEAAFGPGTG PIWLNEIKCR 1001 GNESSLWDCP AKPWSHSDCG HKEDASIQCL PKMTSESHHGTGHPTLTALL 1051 VCGAILLVLL IVFLLWTLKR RQIQRLTVSS RGEVLIHQVQ YQEMDSKADD1101 LDLLKSSGVI QRHTEKENDN L gctttggaat gggtggacac agaatggttc ttcttggaggtgctggatct cctggttgta 60 ID NO: aaaggtttgt ccatctaggt ttctttgttgtggctgtgag ctcacttctc agtgcctctg 120 25 ctgtcactaa cgctcctgga gaaatgaagaaggaactgag actggcgggt ggtgaaaaca 180 actgtagtgg gagagtggaa cttaagatccatgacaagtg gggcacagtg tgcagtaacg 240 gctggagcat gaatgaagtg tccgtggtttgccagcagct gggatgccca acttctatta 300 aagcccttgg atgggctaac tccagcgccggctctggata tatctggatg gacaaagttt 360 cttgtacagg gaatgagtca gctctttgggactgcaaaca tgatgggtgg ggaaagcata 420 actgtaccca tgaaaaagat gctggagtgacctgctcaga tggatctaat ttggagatga 480 gactggtgaa cagtgcgggc caccgatgcttaggaagagt agaaataaag ttccagggaa 540 agtgggggac ggtgtgtgac gacaacttcagcaaagatca cgcttctgtg atttgtaaac 600 agcttggatg tggaagtgcc attagtttctctggctcagc taaattggga gctggttctg 660 gaccaatctg gctcgatgac ctggcatgcaatggaaatga gtcagctctc tgggactgca 720 aacaccgggg atggggcaag cataactgtgaccatgctga ggatgtcggt gtgatttgct 780 tagagggagc agatctgagc ctgagactagtggatggagt gtccagatgt tcaggaagat 840 tggaagtgag attccaagga gaatgggggaccgtgtgtga tgataactgg gatctccggg 900 atgcttctgt ggtgtgcaag caactgggatgtccaactgc catcagtgcc attggtcgag 960 ttaatgccag tgagggatct ggacagatttggcttgacaa catttcatgc gaaggacatg 1020 aggcaactct ttgggagtgt aaacaccaagagtggggaaa gcattactgt catcatagag 1080 aagacgctgg cgtgacatgt tctgatggagcagatctgga acttagactt gtaggtggag 1140 gcagtcgctg tgctggcatt gtggaggtggagattcagaa gctgactggg aagatgtgta 1200 gccgaggctg gacactggca gatgcggatgtggtttgcag acagcttgga tgtggatctg 1260 cgcttcaaac ccaggctaag atctactctaaaactggggc aacaaatacg tggctctttc 1320 ctggatcttg taatggaaat gaaactactttttggcaatg caaaaactgg cagtggggcg 1380 gcctttcctg tgataatttc gaagaagccaaagttacctg ctcaggccac agggaaccca 1440 gactggttgg aggagaaatc ccatgctctggtcgtgtgga aatgaaacac ggagacgtgt 1500 ggggctccgt ctgtgatttt gacttgtctctggaagctgc cagtgtggtg tgcagggaat 1560 tacaatgtgg aacagtcgtc tctatcctagggggagcaca ttttggagaa ggaagtggac 1620 agatctgggg tgaagaattc cagtgtagtggggatgagtc ccatctttca ctatgctcag 1680 tggcgccccc gctagacaga acttgtacccacagcaggga tgtcagcgta gtctgctcac 1740 gatacataga tattcgtctg gcaggcggcgagtcctcctg tgagggaaga gtggagctca 1800 agacactcgg agcctggggt cccctctgcagttctcattg ggacatggaa gatgctcatg 1860 tcttatgtca gcagctgaag tgtggggttgcccaatctat tccagaagga gcacattttg 1920 ggaaaggagc tggtcaggtc tggagtcacatgttccactg cactggaact gaggaacata 1980 taggagattg cctcatgact gctctgggtgcgccgacgtg ttccgaagga caggtggcct 2040 ctgtcatctg ctcaggaaac caatcccagacactattgcc atgtagttca ttgtctccag 2100 tccaaacaac aagctctaca attccaaaggagagtgaagt tccctgcata gcaagtggcc 2160 agcttcgctt ggtaggtgga ggtggtcgctgcgctggaag agtggaggtc taccacgagg 2220 gctcttgggg caccgtctgt gatgacaattgggatatgac tgatgccaat gtggtgtgca 2280 agcagctgga ctgtggcgtg gcaattaacgccactggctc tgcttacttc ggggaaggag 2340 caggagctat ctggctagac gaagtcatctgcactgggaa agagtctcat atttggcagt 2400 gccattcaca tggctgggga cgccataactgcaggcacaa agaagatgca ggtgttatct 2460 gctccgagtt catgtctctg aggctgaccaacgaagccca caaagaaaac tgcacaggtc 2520 gccttgaagt gttttacaat ggtacatggggcagtattgg cagtagcaat atgtctccaa 2580 ccactgtggg ggtggtgtgc cgtcagctgggctgtgcaga caacgggact gtgaaaccca 2640 taccttcaga caagacacca tccaggcccatgtgggtaga tcgtgtgcag tgtccaaaag 2700 gagttgacac tttgtggcag tgcccctcgtcaccttggaa acagagacag gccagcccct 2760 cctcccagga gtcctggatc atctgtgacaacaaaataag actccaggaa gggcatacag 2820 actgttctgg acgtgtggag atctggcacaaaggttcctg gggaacagtg tgtgatgact 2880 cctgggatct taatgatgct aaggttgtatgtaagcagtt gggctgtggc caagctgtga 2940 aggcactaaa agaagcagca tttggtccaggaactgggcc catatggctc aatgaaatta 3000 agtgtagagg gaatgagtct tccctgtgggattgtcctgc caaaccgtgg agtcacagcg 3060 actgtgggca caaagaagat gcttccatccagtgcctccc caaaatgact tcagaatcac 3120 atcatggcac aggtcacccc accctcacggcactcttggt ttgtggagcc attctattgg 3180 tcctcctcat tgtcttcctc ctgtggactctgaagcgacg acagattcag cgacttacag 3240 tttcctcaag aggagaggtc ttgatacatcaagttcagta ccaagagatg gattcaaagg 3300 cggatgatct ggacttgctg aaatcctcggaaaattccaa caattcatat gattttaatg 3360 atgatggact gacatctttg tctaaatatcttcctatttc tggaattaaa aaggggtcat 3420 tcagaggcac actgagaagg aaaatgataatttataatcc actgaggttg gagtttaaga 3480 agccttgaca ggacagccag ctaaatggaacaagagccca ggcaacgcac ggatgaccac 3540 agctgcatct tcatgcagtc ctttgtttcctggaactctg ctgaacctgc aaaaaccata 3600 tttgtgaatg tgaccactta atagagatgggagactttt 3639 1M  G  G  H  R  M  V  L  L  G  G  A  G  S  P  G  C  K  R  F ID NO: 1atgggtggacacagaatggttcttcttggaggtgctggatctcctggttgtaaaaggttt 26 21V  H  L  G  F  F  V  V  A  V  S  S  L  L  S  A  S  A  V  T and 61gtccatctaggtttctttgttgtggctgtgagctcacttctcagtgcctctgctgtcact 27 41N  A  P  G  E  M  K  K  E  L  R  L  A  G  G  E  N  N  C  S 121aacgctcctggagaaatgaagaaggaactgagactggcgggtggtgaaaacaactgtagt 61G  R  V  E  L  K  I  H  D  K  W  G  T  V  C  S  N  G  W  S 181gggagagtggaacttaagatccatgacaagtggggcacagtgtgcagtaacggctggagc 81M  N  E  V  S  V  V  C  Q  Q  L  G  C  P  T  S  I  K  A  L 241atgaatgaagtgtccgtggtttgccagcagctgggatgcccaacttctattaaagccctt 101G  W  A  N  S  S  A  G  S  G  Y  I  W  M  D  K  V  S  C  T 301ggatgggctaactccagcgccggctctggatatatctggatggacaaagtttcttgtaca 121G  N  E  S  A  L  W  D  C  K  H  D  G  W  G  K  H  N  C  T 361gggaatgagtcagctctttgggactgcaaacatgatgggtggggaaagcataactgtacc 141H  E  K  D  A  G  V  T  C  S  D  G  S  N  L  E  M  R  L  V 421catgaaaaagatgctggagtgacctgctcagatggatctaatttggagatgagactggtg 161N  S  A  G  H  R  C  L  G  R  V  E  I  K  F  Q  G  K  W  G 481aacagtgcgggccaccgatgcttaggaagagtagaaataaagttccagggaaagtggggg 181T  V  C  D  D  N  F  S  K  D  H  A  S  V  I  C  K  Q  L  G 541acggtgtgtgacgacaacttcagcaaagatcacgcttctgtgatttgtaaacagcttgga 201C  G  S  A  I  S  F  S  G  S  A  K  L  G  A  G  S  G  P  I 601tgtggaagtgccattagtttctctggctcagctaaattgggagctggttctggaccaatc 221W  L  D  D  L  A  C  N  G  N  E  S  A  L  W  D  C  K  H  R 661tggctcgatgacctggcatgcaatggaaatgagtcagctctctgggactgcaaacaccgg 241G  W  G  K  H  N  C  D  H  A  E  D  V  G  V  I  C  L  E  G 721ggatggggcaagcataactgtgaccatgctgaggatgtcggtgtgatttgcttagaggga 261A  D  L  S  L  R  L  V  D  G  V  S  R  C  S  G  R  L  E  V 781gcagatctgagcctgagactagtggatggagtgtccagatgttcaggaagattggaagtg 281R  F  Q  G  E  W  G  T  V  C  D  D  N  W  D  L  R  D  A  S 841agattccaaggagaatgggggaccgtgtgtgatgataactgggatctccgggatgcttct 301V  V  C  K  Q  L  G  C  P  T  A  I  S  A  I  G  R  V  N  A 901gtggtgtgcaagcaactgggatgtccaactgccatcagtgccattggtcgagttaatgcc 321S  E  G  S  G  Q  I  W  L  D  N  I  S  C  E  G  H  E  A  T 961agtgagggatctggacagatttggcttgacaacatttcatgcgaaggacatgaggcaact 341L  W  E  C  K  H  Q  E  W  G  K  H  Y  C  H  H  R  E  D  A 1021ctttgggagtgtaaacaccaagagtggggaaagcattactgtcatcatagagaagacgct 361G  V  T  C  S  D  G  A  D  L  E  L  R  L  V  G  G  G  S  R 1081ggcgtgacatgttctgatggagcagatctggaacttagacttgtaggtggaggcagtcgc 381C  A  G  I  V  E  V  E  I  Q  K  L  T  G  K  M  C  S  R  G 1141tgtgctggcattgtggaggtggagattcagaagctgactgggaagatgtgtagccgaggc 401W  T  L  A  D  A  D  V  V  C  R  Q  L  G  C  G  S  A  L  Q 1201tggacactggcagatgcggatgtggtttgcagacagcttggatgtggatctgcgcttcaa 421T  Q  A  K  I  Y  S  K  T  G  A  T  N  T  W  L  F  P  G  S 1261acccaggctaagatctactctaaaactggggcaacaaatacgtggctctttcctggatct 441C  N  G  N  E  T  T  F  W  Q  C  K  N  W  Q  W  G  G  L  S 1321tgtaatggaaatgaaactactttttggcaatgcaaaaactggcagtggggcggcctttcc 461C  D  N  F  E  E  A  K  V  T  C  S  G  H  R  E  P  R  L  V 1381tgtgataatttcgaagaagccaaagttacctgctcaggccacagggaacccagactggtt 481G  G  E  I  P  C  S  G  R  V  E  M  K  H  G  D  V  W  G  S 1441ggaggagaaatcccatgctctggtcgtgtggaaatgaaacacggagacgtgtggggctcc 501V  C  D  F  D  L  S  L  E  A  A  S  V  V  C  R  E  L  Q  C 1501gtctgtgattttgacttgtctctggaagctgccagtgtggtgtgcagggaattacaatgt 521G  T  V  V  S  I  L  G  G  A  H  F  G  E  G  S  G  Q  I  W 1561ggaacagtcgtctctatcctagggggagcacattttggagaaggaagtggacagatctgg 541G  E  E  F  Q  C  S  G  D  E  S  H  L  S  L  C  S  V  A  P 1621ggtgaagaattccagtgtagtggggatgagtcccatctttcactatgctcagtggcgccc 561P  L  D  R  T  C  T  H  S  R  D  V  S  V  V  C  S  R  Y  I 1681ccgctagacagaacttgtacccacagcagggatgtcagcgtagtctgctcacgatacata 581D  I  R  L  A  G  G  E  S  S  C  E  G  R  V  E  L  K  T  L 1741gatattcgtctggcaggcggcgagtcctcctgtgagggaagagtggagctcaagacactc 601G  A  W  G  P  L  C  S  S  H  W  D  M  E  D  A  H  V  L  C 1801ggagcctggggtcccctctgcagttctcattgggacatggaagatgctcatgtcttatgt 621Q  Q  L  K  C  G  V  A  Q  S  I  P  E  G  A  H  F  G  K  G 1861cagcagctgaagtgtggggttgcccaatctattccagaaggagcacattttgggaaagga 641A  G  Q  V  W  S  H  M  F  H  C  T  G  T  E  E  H  I  G  D 1921gctggtcaggtctggagtcacatgttccactgcactggaactgaggaacatataggagat 661C  L  M  T  A  L  G  A  P  T  C  S  E  G  Q  V  A  S  V  I 1981tgcctcatgactgctctgggtgcgccgacgtgttccgaaggacaggtggcctctgtcatc 681C  S  G  N  Q  S  Q  T  L  L  P  C  S  S  L  S  P  V  Q  T 2041tgctcaggaaaccaatcccagacactattgccatgtagttcattgtctccagtccaaaca 701T  S  S  T  I  P  K  E  S  E  V  P  C  I  A  S  G  Q  L  R 2101acaagctctacaattccaaaggagagtgaagttccctgcatagcaagtggccagcttcgc 721L  V  G  G  G  G  R  C  A  G  R  V  E  V  Y  H  E  G  S  W 2161ttggtaggtggaggtggtcgctgcgctggaagagtggaggtctaccacgagggctcttgg 741G  T  V  C  D  D  N  W  D  M  T  D  A  N  V  V  C  K  Q  L 2221ggcaccgtctgtgatgacaattgggatatgactgatgccaatgtggtgtgcaagcagctg 761D  C  G  V  A  I  N  A  T  G  S  A  Y  F  G  E  G  A  G  A 2281gactgtggcgtggcaattaacgccactggctctgcttacttcggggaaggagcaggagct 781I  W  L  D  E  V  I  C  T  G  K  E  S  H  I  W  Q  C  H  S 2341atctggctagacgaagtcatctgcactgggaaagagtctcatatttggcagtgccattca 801H  G  W  G  R  H  N  C  R  H  K  E  D  A  G  V  I  C  S  E 2401catggctggggacgccataactgcaggcacaaagaagatgcaggtgttatctgctccgag 821F  M  S  L  R  L  T  N  E  A  H  K  E  N  C  T  G  R  L  E 2461ttcatgtctctgaggctgaccaacgaagcccacaaagaaaactgcacaggtcgccttgaa 841V  F  Y  N  G  T  W  G  S  I  G  S  S  N  M  S  P  T  T  V 2521gtgttttacaatggtacatggggcagtattggcagtagcaatatgtctccaaccactgtg 861G  V  V  C  R  Q  L  G  C  A  D  N  G  T  V  K  P  I  P  S 2581ggggtggtgtgccgtcagctgggctgtgcagacaacgggactgtgaaacccataccttca 881D  K  T  P  S  R  P  M  W  V  D  R  V  Q  C  P  K  G  V  D 2641gacaagacaccatccaggcccatgtgggtagatcgtgtgcagtgtccaaaaggagttgac 901T  L  W  Q  C  P  S  S  P  W  K  Q  R  Q  A  S  P  S  S  Q 2701actttgtggcagtgcccctcgtcaccttggaaacagagacaggccagcccctcctcccag 921E  S  W  I  I  C  D  N  K  I  R  L  Q  E  G  H  T  D  C  S 2761gagtcctggatcatctgtgacaacaaaataagactccaggaagggcatacagactgttct 941G  R  V  E  I  W  H  K  G  S  W  G  T  V  C  D  D  S  W  D 2821ggacgtgtggagatctggcacaaaggttcctggggaacagtgtgtgatgactcctgggat 961L  N  D  A  K  V  V  C  K  Q  L  G  C  G  Q  A  V  K  A  L 2881cttaatgatgctaaggttgtatgtaagcagttgggctgtggccaagctgtgaaggcacta 981K  E  A  A  F  G  P  G  T  G  P  I  W  L  N  E  I  K  C  R 2941aaagaagcagcatttggtccaggaactgggcccatatggctcaatgaaattaagtgtaga 1001G  N  E  S  S  L  W  D  C  P  A  K  P  W  S  H  S  D  C  G 3001gggaatgagtcttccctgtgggattgtcctgccaaaccgtggagtcacagcgactgtggg 1021H  K  E  D  A  S  I  Q  C  L  P  K  M  T  S  E  S  H  H  G 3061cacaaagaagatgcttccatccagtgcctccccaaaatgacttcagaatcacatcatggc 1041T  G  H  P  T  L  T  A  L  L  V  C  G  A  I  L  L  V  L  L 3121acaggtcaccccaccctcacggcactcttggtttgtggagccattctattggtcctcctc 1061I  V  F  L  L  W  T  L  K  R  R  Q  I  Q  R  L  T  V  S  S 3181attgtcttcctcctgtggactctgaagcgacgacagattcagcgacttacagtttcctca 1081R  G  E  V  L  I  H  Q  V  Q  Y  Q  E  M  D  S  K  A  D  D 3241agaggagaggtcttgatacatcaagttcagtaccaagagatggattcaaaggcggatgat 1101L  D  L  L  K  S  S  E  N  S  N  N  S  Y  D  F  N  D  D  G 3301ctggacttgctgaaatcctcggaaaattccaacaattcatatgattttaatgatgatgga 1121L  T  S  L  S  K  Y  L  P  I  S  G  I  K  K  G  S  F  R  G 3361ctgacatctttgtctaaatatcttcctatttctggaattaaaaaggggtcattcagaggc 1141T  L  R  R  K  M  I  I  Y  N  P  L  R  L  E  F  K  K  P 3421acactgagaaggaaaatgataatttataatccactgaggttggagtttaagaagcct 1 MGGHRMVLLGGGAGSPGCKRF VHLGEFVVAV SSLLSASAVT NAPGEMKKEL ID NO: 51 RLAGGENNCSGRVELKIHDK WGTVCSNGWS MNEVSVVCQQ LGCPTSIKAL 27 101 GWANSSAGSG YIWMDKVSCTGNESALWDCK HDGWGKHNCT HEKDAGVTCS 151 DGSNLEMRLV NSAGHRCLGR VEIKFQGKWGTVCDDNFSKD HASVICKQLG 201 CGSAISFSGS AKLGAGSGPI WLDDLACNGN ESALWDCKHRGWGKHNCDHA 251 EDVGVICLEG ADLSLRLVDG VSRCSGRLEV RFQGEWGTVC DDNWDLRDAS301 VVCKQLGCPT AISAIGRVNA SEGSGQIWLD NISCEGHEAT LWECKHQEWG 351KHYCHHREDA GVTCSDGADL ELRLVGGGSR CAGIVEVEIQ KLTGKMCSRG 401 WTLADADVVCRQLGCGSALQ TQAKIYSKTG ATNTWLFPGS CNGNETTFWQ 451 CKNWQWGGLS CDNFEEAKVTCSGHREPRLV GGEIPCSGRV EMKHGDVWGS 501 VCDFDLSLEA ASVVCRELQC GTVVSILGGAHFGEGSGQIW GEEFQCSGDE 551 SHLSLCSVAP PLDRTCTHSR DVSVVCSRYI DIRLAGGESSCEGRVELKTL 601 GAWGPLCSSH WDMEDAHVLC QQLKCGVAQS IPEGAHFGKG AGQVWSHMFH651 CTGTEEHIGD CLMTALGAPT CSEGQVASVI CSGNQSQTLL PCSSLSPVQT 701TSSTIPKESE VPCIASGQLR LVGGGGRCAG RVEVYHEGSW GTVCDDNWDM 751 TDANVVCKQLDCGVAINATG SAYFGEGAGA IWLDEVICTG KESHIWQCHS 801 HGWGRHNCRH KEDAGVICSEFMSLRLTNEA HKENCTGRLE VFYNGTWGSI 851 GSSNMSPTTV GVVCRQLGCA DNGTVKPIPSDKTPSRPMWV DRVQCPKGVD 901 TLWQCPSSPW KQRQASPSSQ ESWIICDNKI RLQEGHTDCSGRVEIWHKGS 951 WGTVCDDSWD LNDAKVVCKQ LGCGQAVKAL KEAAFGPGTG PIWLNEIKCR1001 GNESSLWDCP AKPWSHSDCG HKEDASIQCL PKMTSESHHG TGHPTLTALL 1051VCGAILLVLL IVFLLWTLKR RQIQRLTVSS RGEVLIHQVQ YQEMDSKADD 1101 LDLLKSSENSNNSYDFNDDG LTSLSKYLPI SGIKKGSFRG TLRRKMIIYN 1151 PLRLEFKKP

Example 8 Cloning and Characterization of MARC-145 CD163

A forward primer 5′ simianCD163 (SEQ ID NO: 28)(5′-CACCGGAATGAGCAAACTCAGAATGG-3′ based on human CD163) and a reverseprimer HuCD163-3′Kpn (SEQ ID NO:29)(5′-TGCTCCGGTACCTAGTCCAGGTCTTCATCAAGGTATCTTA-3′) were used to amplifyCD163 cDNA from MARC-145 African Green Monkey kidney cells. Totalcellular RNA was prepared from MARC-145 cells using the RNeasy kit.RT-PCR parameters were the same as described in Example 4. RT-PCRproducts were cloned directionally into the pcDNA3.1D/V5/His/TOPO vectoraccording to the manufacturer's instruction. Several clones containinglarge inserts were analyzed. Clone #25 was designated“pcDNA3.1D-MARC-CD163v2”. This novel CD163 cDNA from MARC-145 cells is1116 amino acids in length. When compared to the sequences in GenBankdatabase, the MARC-145 CD163 amino acid sequence is 96.3% identical tohuman CD163 (Genbank Z22968), 84.7% identical to pig CD163 (GenbankAJ311716), and 73.9% identical to mouse CD163 (Genbank AF274883).

SEQUENCE ID NOatgagcaaac tcagaatggt gctacttgaa gactctggat ctgctgacgt cagaagacat 60SEQ ID tttgtcaact tgagtccctt cactattgct gtggtcttac ttctccgtgc ctgttttgtc120 NO: 30accagttctc ttggaggaac aaccaaggag ctgaggctag tggatggtga aaacaagtgt 180agtgggagag tggaagtgaa aatccaggag gagtggggaa cggtgtgtaa taatggctgg 240agcatggaag cagtctctgt gatttgtaac cagctgggat gtccaactgc tatcaaagcc 300actggatggg ctaattccag tgcaggttct ggacgcattt ggatggatca tgtttcttgt 360cgtgggaatg agtcagctct ttgggactgc aaacatgatg gatggggaaa gcatagtaac 420tgtactcacc aacaagatgc tggagtgact tgctcagatg gatccgattt ggaaatgagg 480ctgacgaatg gagggaatat gtgttctgga agaatagaga tcaaattcca aggacagtgg 540ggaacagtgt gtgatgataa cttcaacatc aatcatgcat ctgtggtttg taaacaactt 600gaatgtggaa gtgctgtcag tttctctggt tcagctaatt ttggagaagg ctctggacca 660atctggtttg atgatcttat atgcaacgga aatgagtcag ctctctggaa ctgcaaacat 720caaggatggg gaaagcataa ctgtgatcat gctgaggatg ctggagtgat ttgctcaaag 780ggagcagatc tgagcctgag actggtagat ggagtcactg aatgttcagg aagattagaa 840gtgagattcc aaggagaatg ggggacaata tgtgatgacg gctgggacag tcatgatgct 900gctgtggcat gcaagcaact gggatgtcca actgctatca ccgccattgg tcgagttaac 960gccagtgagg gatttggaca catctggctt gacagtgttt cttgccaggg acatgaacct 1020gcggtctggc aatgtaaaca ccatgaatgg ggaaagcatt attgcaatca caatgaagat 1080gctggcgtaa catgttctga tggatcagat ctggagctaa gacttagagg tggaggcagc 1140cgctgtgctg ggacagttga ggtggagatt cagagactgt tagggaaggt gtgtgacaga 1200ggctggggac tgaaagaagc tgatgtggtt tgcaggcagc tgggatgtgg atctgcactc 1260aaaacatcct atcaagtata ctccaaaatc caggcaacaa acatgtggct gtttctaagt 1320agctgtaacg gaaatgaaac ttctctttgg gactgcaaga actggcaatg gggtggactt 1380acctgtgatc actatgaaga agccaaaatt acctgctcag cccacaggga acccagactg 1440gttggaggag acattccctg ttctggacgc gttgaagtga agcatggtga cacatggggc 1500tccgtctgtg attcggattt ctctctggaa gctgccagcg ttctatgcag ggaattacag 1560tgtggcacag tcgtctctat cctgggggga gctcactttg gagagggaaa tggacagatc 1620tgggctgaag aattccagtg tgagggacat gagtcccatc tttcactctg cccagtagca 1680ccccgcccag aaggaacttg tagccacagc agggatgttg gagtagtctg ctcaagatac 1740acagaaattc gcttggtgaa tggcaagacc ccatgtgagg gcagagtgga gctcaaaacg 1800cttaatgcct ggggatccct ctgcaactct cactgggaca tagaagatgc ccacgttctt 1860tgccaacaac ttaaatgtgg agttgccctt tctaccccag gaggagcaca ttttggaaaa 1920ggaaatggtc aggtctggag gcatatgttt cactgcactg ggactgagca gcacatggga 1980gattgtcctg taactgctct gggtgcttca ctatgtcctt cagggcaagt ggcctctgta 2040atttgctcag gaaaccagtc ccaaacactg tcctcgtgca attcatcatc tctgggccca 2100acaaggccta ccattccaga agaaagtgct gtggcctgca tagagagtgg tcaacttcgc 2160ttggtaaatg gaggaggtcg ctgtgctggg agagtagaga tttatcatga gggctcctgg 2220ggcaccatct gtgatgacag ctgggacctg agcgatgccc acgtggtgtg cagacagctg 2280ggctgtggag aggccattaa tgccactggt tctgctcatt ttggagaagg aacagggccc 2340atctggctgg atgagatgaa atgcaatgga aaagaatccc gcatttggca gtgccattca 2400catggctggg ggcagcaaaa ctgcaggcac aaggaggatg caggagttat ctgctcagag 2460ttcatgtctc tgagactgac cagtgaagcc agcagagagg cctgtgcagg gcgtctagaa 2520gttttttaca acggagcttg gggcagtgtt ggcaggagta acatgtctga aaccactgtg 2580ggtgtggtgt gcaggcagct gggctgtgca gacaaaggga aaatcaaccc tgcatcttta 2640gacaaggcca tgtccattcc catgtgggtg gacaatgttc agtgtccaaa aggacctgac 2700acgctgtggc agtgcccatc atctccatgg gagaagagac tggccaggcc ctcggaggag 2760acctggatca catgtgacaa caagatgaga ctacaagaag gacccacttc ctgttctgga 2820cgtgtggaga tctggcacgg aggttcctgg gggacagtgt gtgatgactc ctgggacttg 2880aacgatgctc aggtggtgtg tcaacaactt ggctgtggtc cagctttgaa agcattcaaa 2940gaagcagagt ttggtcaggg gactggaccc atatggctca atgaagtgaa gtgcaaaggg 3000aatgagtctt ccttgtggga ttgtcctgcc agacgctggg gccacagtga gtgtggacac 3060aaggaagacg ctgcagtgaa ttgcacagat atttcaacga acaaaacccc acaaaaagcc 3120acaacaggtc agtcatccct tattgcagtc ggaatccttg gagttgttct cttggtcatt 3180ttcgtcgcat tattcttgac tcaaaagcga agacagagac agcggcttac agtttcctca 3240agaggagaga acttagtcca ccaaattcaa taccgggaga tgaattcttg cctgaatgca 3300gatgatctgg acctaatgaa ttcctcagga ggccattctg aggcacactg aaaaggaaaa 3360tgggaattta taacccagtg agccttgaag ataccttgat gaagacctgg acta 3414 1M  S  K  L  R  M  V  L  L  E  D  S  G  S  A  D  V  R  R  H SEQ ID 1atgagcaaactcagaatggtgctacttgaagactctggatctgctgacgtcagaagacat NO: 31 21F  V  N  L  S  P  F  T  I  A  V  V  L  L  L  R  A  C  F  V and 32 61tttgtcaacttgagtcccttcactattgctgtggtcttacttctccgtgcctgttttgtc 41T  S  S  L  G  G  T  T  K  E  L  R  L  V  D  G  E  N  K  C 121accagttctcttggaggaacaaccaaggagctgaggctagtggatggtgaaaacaagtgt 61S  G  R  V  E  V  K  I  Q  E  E  W  G  T  V  C  N  N  G  W 181agtgggagagtggaagtgaaaatccaggaggagtggggaacggtgtgtaataatggctgg 81S  M  E  A  V  S  V  I  C  N  Q  L  G  C  P  T  A  I  K  A 241agcatggaagcagtctctgtgatttgtaaccagctgggatgtccaactgctatcaaagcc 101T  G  W  A  N  S  S  A  G  S  G  R  I  W  M  D  H  V  S  C 301actggatgggctaattccagtgcaggttctggacgcatttggatggatcatgtttcttgt 121R  G  N  E  S  A  L  W  D  C  K  H  D  G  W  G  K  H  S  N 361cgtgggaatgagtcagctctttgggactgcaaacatgatggatggggaaagcatagtaac 141C  T  H  Q  Q  D  A  G  V  T  C  S  D  G  S  D  L  E  M  R 421tgtactcaccaacaagatgctggagtgacttgctcagatggatccgatttggaaatgagg 161L  T  N  G  G  N  M  C  S  G  R  I  E  I  K  F  Q  G  Q  W 481ctgacgaatggagggaatatgtgttctggaagaatagagatcaaattccaaggacagtgg 181G  T  V  C  D  D  N  F  N  I  N  H  A  S  V  V  C  K  Q  L 541ggaacagtgtgtgatgataacttcaacatcaatcatgcatctgtggtttgtaaacaactt 201E  C  G  S  A  V  S  F  S  G  S  A  N  F  G  E  G  S  G  P 601gaatgtggaagtgctgtcagtttctctggttcagctaattttggagaaggctctggacca 221I  W  F  D  D  L  I  C  N  G  N  E  S  A  L  W  N  C  K  H 661atctggtttgatgatcttatatgcaacggaaatgagtcagctctctggaactgcaaacat 241Q  G  W  G  K  H  N  C  D  H  A  E  D  A  G  V  I  C  S  K 721caaggatggggaaagcataactgtgatcatgctgaggatgctggagtgatttgctcaaag 261G  A  D  L  S  L  R  L  V  D  G  V  T  E  C  S  G  R  L  E 781ggagcagatctgagcctgagactggtagatggagtcactgaatgttcaggaagattagaa 281V  R  F  Q  G  E  W  G  T  I  C  D  D  G  W  D  S  H  D  A 841gtgagattccaaggagaatgggggacaatatgtgatgacggctgggacagtcatgatgct 301A  V  A  C  K  Q  L  G  C  P  T  A  I  T  A  I  G  R  V  N 901gctgtggcatgcaagcaactgggatgtccaactgctatcaccgccattggtcgagttaac 321A  S  E  G  F  G  H  I  W  L  D  S  V  S  C  Q  G  H  E  P 961gccagtgagggatttggacacatctggcttgacagtgtttcttgccagggacatgaacct 341A  V  W  Q  C  K  H  H  E  W  G  K  H  Y  C  N  H  N  E  D 1021gcggtctggcaatgtaaacaccatgaatggggaaagcattattgcaatcacaatgaagat 361A  G  V  T  C  S  D  G  S  D  L  E  L  R  L  R  G  G  G  S 1081gctggcgtaacatgttctgatggatcagatctggagctaagacttagaggtggaggcagc 381R  C  A  G  T  V  E  V  E  I  Q  R  L  L  G  K  V  C  D  R 1141cgctgtgctgggacagttgaggtggagattcagagactgttagggaaggtgtgtgacaga 401G  W  G  L  K  E  A  D  V  V  C  R  Q  L  G  C  G  S  A  L 1201ggctggggactgaaagaagctgatgtggtttgcaggcagctgggatgtggatctgcactc 421K  T  S  Y  Q  V  Y  S  K  I  Q  A  T  N  M  W  L  F  L  S 1261aaaacatcctatcaagtatactccaaaatccaggcaacaaacatgtggctgtttctaagt 441S  C  N  G  N  E  T  S  L  W  D  C  K  N  W  Q  W  G  G  L 1321agctgtaacggaaatgaaacttctctttgggactgcaagaactggcaatggggtggactt 461T  C  D  H  Y  E  E  A  K  I  T  C  S  A  H  R  E  P  R  L 1381acctgtgatcactatgaagaagccaaaattacctgctcagcccacagggaacccagactg 481V  G  G  D  I  P  C  S  G  R  V  E  V  K  H  G  D  T  W  G 1441gttggaggagacattccctgttctggacgcgttgaagtgaagcatggtgacacatggggc 501S  V  C  D  S  D  F  S  L  E  A  A  S  V  L  C  R  E  L  Q 1501tccgtctgtgattcggatttctctctggaagctgccagcgttctatgcagggaattacag 521C  G  T  V  V  S  I  L  G  G  A  H  F  G  E  G  N  G  Q  I 1561tgtggcacagtcgtctctatcctggggggagctcactttggagagggaaatggacagatc 541W  A  E  E  F  Q  C  E  G  H  E  S  H  L  S  L  C  P  V  A 1621tgggctgaagaattccagtgtgagggacatgagtcccatctttcactctgcccagtagca 561P  R  P  E  G  T  C  S  H  S  R  D  V  G  V  V  C  S  R  Y 1681ccccgcccagaaggaacttgtagccacagcagggatgttggagtagtctgctcaagatac 581T  E  I  R  L  V  N  G  K  T  P  C  E  G  R  V  E  L  K  T 1741acagaaattcgcttggtgaatggcaagaccccatgtgagggcagagtggagctcaaaacg 601L  N  A  W  G  S  L  C  N  S  H  W  D  I  E  D  A  H  V  L 1801cttaatgcctggggatccctctgcaactctcactgggacatagaagatgcccacgttctt 621C  Q  Q  L  K  C  G  V  A  L  S  T  P  G  G  A  H  F  G  K 1861tgccaacaacttaaatgtggagttgccctttctaccccaggaggagcacattttggaaaa 641G  N  G  Q  V  W  R  H  M  F  H  C  T  G  T  E  Q  H  M  G 1921ggaaatggtcaggtctggaggcatatgtttcactgcactgggactgagcagcacatggga 661D  C  P  V  T  A  L  G  A  S  L  C  P  S  G  Q  V  A  S  V 1981gattgtcctgtaactgctctgggtgcttcactatgtccttcagggcaagtggcctctgta 681I  C  S  G  N  Q  S  Q  T  L  S  S  C  N  S  S  S  L  G  P 2041atttgctcaggaaaccagtcccaaacactgtcctcgtgcaattcatcatctctgggccca 701T  R  P  T  I  P  E  E  S  A  V  A  C  I  E  S  G  Q  L  R 2101acaaggcctaccattccagaagaaagtgctgtggcctgcatagagagtggtcaacttcgc 721L  V  N  G  G  G  R  C  A  G  R  V  E  I  Y  H  E  G  S  W 2161ttggtaaatggaggaggtcgctgtgctgggagagtagagatttatcatgagggctcctgg 741G  T  I  C  D  D  S  W  D  L  S  D  A  H  V  V  C  R  Q  L 2221ggcaccatctgtgatgacagctgggacctgagcgatgcccacgtggtgtgcagacagctg 761G  C  G  E  A  I  N  A  T  G  S  A  H  F  G  E  G  T  G  P 2281ggctgtggagaggccattaatgccactggttctgctcattttggagaaggaacagggccc 781I  W  L  D  E  M  K  C  N  G  K  E  S  R  I  W  Q  C  H  S 2341atctggctggatgagatgaaatgcaatggaaaagaatcccgcatttggcagtgccattca 801H  G  W  G  Q  Q  N  C  R  H  K  E  D  A  G  V  I  C  S  E 2401catggctgggggcagcaaaactgcaggcacaaggaggatgcaggagttatctgctcagag 821F  M  S  L  R  L  T  S  E  A  S  R  E  A  C  A  G  R  L  E 2461ttcatgtctctgagactgaccagtgaagccagcagagaggcctgtgcagggcgtctagaa 841V  F  Y  N  G  A  W  G  S  V  G  R  S  N  M  S  E  T  T  V 2521gttttttacaacggagcttggggcagtgttggcaggagtaacatgtctgaaaccactgtg 861G  V  V  C  R  Q  L  G  C  A  D  K  G  K  I  N  P  A  S  L 2581ggtgtggtgtgcaggcagctgggctgtgcagacaaagggaaaatcaaccctgcatcttta 881D  K  A  M  S  I  P  M  W  V  D  N  V  Q  C  P  K  G  P  D 2641gacaaggccatgtccattcccatgtgggtggacaatgttcagtgtccaaaaggacctgac 901T  L  W  Q  C  P  S  S  P  W  E  K  R  L  A  R  P  S  E  E 2701acgctgtggcagtgcccatcatctccatgggagaagagactggccaggccctcggaggag 921T  W  I  T  C  D  N  K  M  R  L  Q  E  G  P  T  S  C  S  G 2761acctggatcacatgtgacaacaagatgagactacaagaaggacccacttcctgttctgga 941R  V  E  I  W  H  G  G  S  W  G  T  V  C  D  D  S  W  D  L 2821cgtgtggagatctggcacggaggttcctgggggacagtgtgtgatgactcctgggacttg 961N  D  A  Q  V  V  C  Q  Q  L  G  C  G  P  A  L  K  A  F  K 2881aacgatgctcaggtggtgtgtcaacaacttggctgtggtccagctttgaaagcattcaaa 981E  A  E  F  G  Q  G  T  G  P  I  W  L  N  E  V  K  C  K  G 2941gaagcagagtttggtcaggggactggacccatatggctcaatgaagtgaagtgcaaaggg 1001N  E  S  S  L  W  D  C  P  A  R  R  W  G  H  S  E  C  G  H 3001aatgagtcttccttgtgggattgtcctgccagacgctggggccacagtgagtgtggacac 1021K  E  D  A  A  V  N  C  T  D  I  S  T  N  K  T  P  Q  K  A 3061aaggaagacgctgcagtgaattgcacagatatttcaacgaacaaaaccccacaaaaagcc 1041T  T  G  Q  S  S  L  I  A  V  G  I  L  G  V  V  L  L  V  I 3121acaacaggtcagtcatcccttattgcagtcggaatccttggagttgttctcttggtcatt 1061F  V  A  L  F  L  T  Q  K  R  R  Q  R  Q  R  L  T  V  S  S 3181ttcgtcgcattattcttgactcaaaagcgaagacagagacagcggcttacagtttcctca 1081R  G  E  N  L  V  H  Q  I  Q  Y  R  E  M  N  S  C  L  N  A 3241agaggagagaacttagtccaccaaattcaataccgggagatgaattcttgcctgaatgca 1101D  D  L  D  L  M  N  S  S  G  G  H  S  E  A  H 3301gatgatctggacctaatgaattcctcaggaggccattctgaggcacac 1MSKLRMVLLE DSGSADVRRH FVNLSPFTIA VVLLLRACFV TSSLGGTTKE SEQ ID 51LRLVDGENKC SGRVEVKIQE EWGTVCNNGW SMEAVSVICN QLGCPTAIKA NO: 32 101TGWANSSAGS GRIWMDHVSC RGNESALWDC KHDGWGKHSN CTHQQDAGVT 151CSDGSDLEMR LTNGGNMCSG RIEIKFQGQW GTVCDDNFNI NHASVVCKQL 201ECGSAVSFSG SANFGEGSGP IWFDDLICNG NESALWNCKH QGWGKHNCDH 251AEDAGVICSK GADLSLRLVD GVTECSGRLE VRFQGEWGTI CDDGWDSHDA 301AVACKQLGCP TAITAIGRVN ASEGFGHIWL DSVSCQGHEP AVWQCKHHEW 351GKHYCNHNED AGVTCSDGSD LELRLRGGGS RCAGTVEVEI QRLLGKVCDR 401GWGLKEADVV CRQLGCGSAL KTSYQVYSKI QATNMWLFLS SCNGNETSLW 451DCKNWQWGGL TCDHYEEAKI TCSAHREPRL VGGDIPCSGR VEVKHGDTWG 501SVCDSDFSLE AASVLCRELQ CGTVVSILGG AHFGEGNGQI WAEEFQCEGH 551ESHLSLCPVA PRPEGTCSHS RDVGVVCSRY TEIRLVNGKT PCEGRVELKT 601LNAWGSLCNS HWDIEDAHVL CQQLKCGVAL STPGGAHFGK GNGQVWRHMF 651HCTGTEQHMG DCPVTALGAS LCPSGQVASV ICSGNQSQTL SSCNSSSLGP 701TRPTIPEESA VACIESGQLR LVNGGGRCAG RVEIYHEGSW GTICDDSWDL 751SDAHVVCRQL GCGEAINATG SAHFGEGTGP IWLDEMKCNG KESRIWQCHS 801HGWGQQNCRH KEDAGVICSE FMSLRLTSEA SREACAGRLE VFYNGAWGSV 851GRSNMSETTV GVVCRQLGCA DKGKINPASL DKAMSIPMWV DNVQCPKGPD 901TLWQCPSSPW EKRLARPSEE TWITCDNKMR LQEGPTSCSG RVEIWHGGSW 951GTVCDDSWDL NDAQVVCQQL GCGPALKAFK EAEFGQGTGP IWLNEVKCKG 1001NESSLWDCPA RRWGHSECGH KEDAAVNCTD ISTNKTPQKA TTGQSSLIAV 1051GILGVVLLVI FVALFLTQKR RQRQRLTVSS RGENLVHQIQ YREMNSCLNA 1101DDLDLMNSSG GHSEAH

Example 9 Cloning and Characterization of Simian CD163 from Vero Cells

A forward primer 5′ simianCD163 (SEQ ID NO: 28)(5′-CACCGGAATGAGCAAACTCAGAATGG-3′ based on human CD163) and a reverseprimer HuCD163-3′Kpn (SEQ ID NO:29)(5′-TGCTCCGGTACCTAGTCCAGGTCTTCATCAAGGTATCTTA-3′) were used to amplifyCD163 cDNA from Vero cells. Total cellular RNA was prepared from Verocells using the RNeasy kit. RT-PCR parameters were the same as describedin Example 4. RT-PCR products were cloned directionally into thepcDNA3.1D/V5/His/TOPO vector according to the manufacturer'sinstruction. Eight clones containing large inserts were sequenced, andsix discreet splicing patterns were found. These patterns are depictedgraphically in FIG. 17.

The six splicing variants differ in the presence or absence of threeexons, designated E6, E105, and E83. Omissions of E6 or E105 do notchange the reading frame, whereas omission of E83 does. Patterns similarto v2 and/or v3 were also seen in porcine, murine, human, and MARC-145monkey cells. Patterns v4 and v5 lack the 105-nucleotide exon thatencodes the hydrophobic transmembrane region. These cDNAs were unable torender BHK cells permissive to PRRSV infection in a transienttransfection assay, probably because the CD163 is secreted rather thanremaining membrane bound. Although CD163 molecules lacking atransmembrane region appear to be non-functional as cellularpermissivity factors, it is possible that they may have utility eitherin direct virus neutralization (similar to neutralizing antibodies), oras an immunogen for the induction of anti-CD163 antibodies which wouldblock viral infection in the host animal.

The longest splice variant, v7, contains all three of the exons E6,E105, and E83. This novel CD163 cDNA from Vero cells encodes apolypeptide that is 1153 amino acids in length. When compared to thesequences in Genbank database, the Vero CD163v7 amino acid sequence is95.4% identical to human CD163 (Genbank Z22968), 83.7% identical to pigCD163 (Genbank AJ311716), and 72.1% identical to mouse CD163 (GenbankAF274883). The nucleotide and amino acid sequences of the six splicevariants found in Vero cells are provided below (SEQ ID NOS:33-44).

SEQUENCE ID NO 1M  S  K  L  R  M  V  L  L  E  D  S  G  S  A  D  V  R  R  H SEQ ID 1ATGAGCAAACTCAGAATGGTGCTACTTGAAGACTCTGGATCTGCTGACGTCAGAAGACAT NO: 33 21F  V  N  L  S  P  F  T  I  A  V  V  L  L  L  R  A  C  F  V and 34 61TTTGTCAACTTGAGTCCCTTCACTATTGCTGTGGTCTTACTTCTCCGTGCCTGTTTTGTC 41T  S  S  L  G  G  T  T  K  E  L  R  L  V  D  G  E  N  K  C 121ACCAGTTCTCTTGGAGGAACAACCAAGGAGCTGAGGCTAGTGGATGGTGAAAACAAGTGT 61S  G  R  V  E  V  K  I  Q  E  E  W  G  T  V  C  N  N  G  W 181AGTGGGAGAGTGGAAGTGAAAATCCAGGAGGAGTGGGGAACGGTGTGTAATAATGGCTGG 81S  M  E  A  V  S  V  I  C  N  Q  L  G  C  P  T  A  I  K  A 241AGCATGGAAGCAGTCTCTGTGATTTGTAACCAGCTGGGATGTCCAACTGCTATCAAAGCC 101T  G  W  A  N  S  S  A  G  S  G  R  I  W  M  D  H  V  S  C 301ACTGGATGGGCTAATTCCAGTGCAGGTTCTGGACGCATTTGGATGGATCATGTTTCTTGT 121R  G  N  E  S  A  L  W  D  C  K  H  D  G  W  G  K  H  S  N 361CGTGGGAATGAGTCAGCTCTTTGGGACTGCAAACATGATGGATGGGGAAAGCATAGTAAC 141C  T  H  Q  Q  D  A  G  V  T  C  S  D  G  S  D  L  E  M  R 421TGTACTCACCAACAAGATGCTGGAGTAACTTGCTCAGATGGATCCGATTTGGAAATGAGG 161L  T  N  G  G  N  M  C  S  G  R  I  E  I  K  F  Q  G  Q  W 481CTGACGAATGGAGGGAATATGTGTTCTGGAAGAATAGAGATCAAATTCCAAGGACAGTGG 181G  T  V  C  D  D  N  F  N  I  N  H  A  S  V  V  C  K  Q  L 541GGAACAGTGTGTGATGATAACTTCAACATCAATCATGCATCTGTGGTTTGTAAACAACTT 201E  C  G  S  A  V  S  F  S  G  S  A  N  F  G  E  G  S  G  P 601GAATGTGGAAGTGCTGTCAGTTTCTCTGGTTCAGCTAATTTTGGAGAAGGCTCTGGACCA 221I  W  F  D  D  L  I  C  N  G  N  E  S  A  L  W  N  C  K  H 661ATCTGGTTTGATGATCTTATATGCAACGGAAATGAGTCAGCTCTCTGGAACTGCAAACAT 241Q  G  W  G  K  H  N  C  D  H  A  E  D  A  G  V  I  C  S  K 721CAAGGATGGGGAAAGCATAACTGTGATCATGCTGAGGATGCTGGAGTGATTTGCTCAAAG 261G  A  D  L  S  L  R  L  V  D  G  V  T  E  C  S  G  R  L  E 781GGAGCAGATCTGAGCCTGAGACTGGTAGATGGAGTCACTGAATGTTCAGGAAGATTAGAA 281V  R  F  Q  G  E  W  G  T  I  C  D  D  G  W  D  S  H  D  A 841GTGAGATTCCAAGGAGAATGGGGGACAATATGTGATGACGGCTGGGACAGTCATGATGCT 301A  V  A  C  K  Q  L  G  C  P  T  A  I  T  A  I  G  R  V  N 901GCTGTGGCATGCAAGCAACTGGGATGTCCAACTGCTATCACCGCCATTGGTCGAGTTAAC 321A  S  E  G  F  G  H  I  W  L  D  S  V  S  C  Q  G  H  E  P 961GCCAGTGAGGGATTTGGACACATCTGGCTTGACAGTGTTTCTTGCCAGGGACATGAACCT 341A  V  W  Q  C  K  H  H  E  W  G  K  H  Y  C  N  H  N  E  D 1021GCGGTCTGGCAATGTAAACACCATGAATGGGGAAAGCATTATTGCAATCACAATGAAGAT 361A  G  V  T  C  S  D  G  S  G  L  E  L  R  L  R  G  G  G  S 1081GCTGGCGTAACATGTTCTGATGGATCAGGTCTGGAGCTAAGACTTAGAGGTGGAGGCAGC 381R  C  A  G  T  V  E  V  E  I  Q  R  L  L  G  K  V  C  D  R 1141CGCTGTGCTGGGACAGTTGAGGTGGAGATTCAGAGACTGTTAGGGAAGGTGTGTGACAGA 401G  W  G  L  K  E  A  D  V  V  C  R  Q  L  G  C  G  S  A  L 1201GGCTGGGGACTGAAAGAAGCTGATGTGGTTTGCAGGCAGCTGGGATGTGGATCTGCACTC 421K  T  S  Y  Q  V  Y  S  K  I  Q  A  T  N  M  W  L  F  L  S 1261AAAACATCCTATCAAGTATACTCCAAAATCCAGGCAACAAACATGTGGCTGTTTCTAAGT 441S  C  N  G  N  E  T  S  L  W  D  C  K  N  W  Q  W  G  G  L 1321AGCTGTAACGGAAATGAAACTTCTCTTTGGGACTGCAAGAACTGGCAATGGGGTGGACTT 461T  C  D  H  Y  E  E  A  K  I  T  C  S  A  H  R  E  P  R  L 1381ACCTGTGATCACTATGAAGAAGCCAAAATTACCTGCTCAGCCCACAGGGAACCCAGACTG 481V  G  G  D  I  P  C  S  G  R  V  E  V  K  H  G  D  T  W  G 1441GTTGGAGGAGACATTCCCTGTTCTGGACGCGTTGAAGTGAAGCATGGTGACACATGGGGC 501S  V  C  D  S  D  F  S  L  E  A  A  S  V  L  C  R  E  L  Q 1501TCCGTCTGTGATTCGGATTTCTCTCTGGAAGCTGCCAGCGTTCTATGCAGGGAATTACAG 521C  G  T  V  V  S  I  L  G  G  A  H  F  G  E  G  N  G  Q  I 1561TGTGGCACAGTCGTCTCTATCCTGGGGGGAGCTCACTTTGGAGAGGGAAATGGACAGATC 541W  T  E  E  F  Q  C  E  G  H  E  S  H  L  S  L  C  P  V  A 1621TGGACTGAAGAATTCCAGTGTGAGGGACATGAGTCCCATCTTTCACTCTGCCCAGTAGCA 561P  R  P  E  G  T  C  S  H  S  R  D  V  G  V  V  C  S  R  Y 1681CCCCGCCCAGAAGGAACTTGTAGCCACAGCAGGGATGTTGGAGTAGTCTGCTCAAGATAC 581T  E  I  R  L  V  N  G  K  T  P  C  E  G  R  V  E  L  K  T 1741ACAGAAATTCGCTTGGTGAATGGCAAGACCCCATGTGAGGGCAGAGTGGAGCTCAAAACG 601L  N  A  W  G  S  L  C  N  S  H  W  D  I  E  D  A  H  V  L 1801CTTAATGCCTGGGGATCCCTCTGCAACTCTCACTGGGACATAGAAGATGCCCACGTTCTT 621C  Q  Q  L  K  C  G  V  A  L  S  T  P  G  G  A  H  F  G  K 1861TGCCAACAACTTAAATGTGGAGTTGCCCTTTCTACCCCAGGAGGAGCACATTTTGGAAAA 641G  N  G  Q  V  W  R  H  M  F  H  C  T  G  T  E  Q  H  M  G 1921GGAAATGGTCAGGTCTGGAGGCATATGTTTCACTGCACTGGGACTGAGCAGCACATGGGA 661D  C  P  V  T  A  L  G  A  S  L  C  P  S  G  Q  V  A  S  V 1981GATTGTCCTGTAACTGCTCTGGGTGCTTCACTATGTCCTTCAGGGCAAGTGGCCTCTGTA 681I  C  S  G  N  Q  S  Q  T  L  S  S  R  N  S  S  S  L  G  P 2041ATTTGCTCAGGAAACCAGTCCCAAACACTGTCCTCGCGCAATTCATCATCTCTGGGCCCA 701T  R  P  T  I  P  E  E  S  A  V  A  C  I  E  S  G  Q  L  R 2101ACAAGGCCTACCATTCCAGAAGAAAGTGCTGTGGCCTGCATAGAGAGTGGTCAACTTCGC 721L  V  N  G  G  G  R  C  A  G  R  V  E  I  Y  H  E  G  S  W 2161TTGGTAAATGGAGGAGGTCGCTGTGCTGGGAGAGTAGAGATTTATCATGAGGGCTCCTGG 741G  T  I  C  D  D  S  W  D  L  S  D  A  H  V  V  C  R  Q  L 2221GGCACCATCTGTGATGACAGCTGGGACCTGAGCGATGCCCACGTGGTGTGCAGACAGCTG 761G  C  G  E  A  I  N  A  T  G  S  A  H  F  G  E  G  T  G  P 2281GGCTGTGGAGAGGCCATTAATGCCACTGGTTCTGCTCATTTTGGAGAAGGAACAGGGCCC 781I  W  L  D  E  M  K  C  N  G  K  E  S  R  I  W  Q  C  H  S 2341ATCTGGCTGGATGAGATGAAATGCAATGGAAAAGAATCCCGCATTTGGCAGTGCCATTCA 801H  G  W  G  Q  Q  N  C  R  H  K  E  D  A  G  V  I  C  S  E 2401CATGGCTGGGGGCAGCAAAACTGCAGGCACAAGGAGGATGCAGGAGTTATCTGCTCAGAG 821F  M  S  L  R  L  T  S  E  A  S  R  E  A  C  A  G  R  L  E 2461TTCATGTCTCTGAGACTGACCAGTGAAGCCAGCAGAGAGGCCTGTGCAGGGCGTCTAGAA 841V  F  Y  N  G  A  W  G  S  V  G  R  S  N  M  S  E  T  T  V 2521GTTTTTTACAACGGAGCTTGGGGCAGTGTTGGCAGGAGTAACATGTCTGAAACCACTGTG 861G  V  V  C  R  Q  L  G  C  A  D  K  G  K  I  N  S  A  S  L 2581GGTGTAGTGTGCAGGCAGCTGGGCTGTGCAGACAAAGGGAAAATCAACTCTGCATCTTTA 881D  K  A  M  S  I  P  M  W  V  D  N  V  Q  C  P  K  G  P  D 2641GACAAGGCCATGTCCATTCCCATGTGGGTGGACAATGTTCAGTGTCCAAAAGGACCTGAC 901T  L  W  Q  C  P  S  S  P  W  E  K  R  L  A  R  P  S  E  E 2701ACGCTGTGGCAGTGCCCATCATCTCCATGGGAGAAGAGACTGGCCAGGCCCTCGGAGGAG 921T  W  I  T  C  D  N  K  M  R  L  Q  E  G  P  T  S  C  S  G 2761ACCTGGATCACATGTGACAACAAGATGAGACTACAAGAAGGACCCACTTCCTGTTCTGGA 941R  V  E  I  W  H  G  G  S  W  G  T  V  C  D  D  S  W  D  L 2821CGTGTGGAGATCTGGCACGGAGGTTCCTGGGGGACAGTGTGTGATGACTCCTGGGACTTG 961N  D  A  Q  V  V  C  Q  Q  L  G  C  G  P  A  L  K  A  F  K 2881AACGATGCTCAGGTGGTGTGTCAACAACTTGGCTGTGGTCCAGCTTTGAAAGCATTCAAA 981E  A  E  F  G  Q  G  T  G  P  I  W  L  N  E  V  K  C  E  G 2941GAAGCAGAGTTTGGTCAGGGGACTGGACCCATATGGCTCAATGAAGTGAAGTGCGAAGGG 1001N  E  S  S  L  W  D  C  P  A  R  R  W  G  H  S  E  C  G  H 3001AATGAGTCTTCCTTGTGGGATTGTCCTGCCAGACGCTGGGGCCACAGTGAGTGTGGACAC 1021K  E  D  A  A  V  N  C  T  D  I  S  T  R  K  T  P  Q  K  A 3061AAGGAAGACGCTGCAGTGAATTGCACAGATATTTCAACGCGCAAAACCCCACAAAAAGCC 1041T  T  G  Q  S  S  L  I  A  V  G  I  L  G  V  V  L  L  A  I 3121ACAACAGGTCAGTCATCCCTTATTGCAGTCGGAATCCTTGGAGTTGTTCTCTTGGCCATT 1061F  V  A  L  F  L  T  Q  K  R  R  Q  R  Q  R  L  T  V  S  S 3181TTCGTCGCATTATTCTTGACTCAAAAGCGAAGACAGAGACAGCGGCTTACAGTTTCCTCA 1081R  G  E  N  L  V  H  Q  I  Q  Y  R  E  M  N  S  C  L  N  A 3241AGAGGAGAGAACTTAGTCCACCAAATTCAATACCGGGAGATGAATTCTTGCCTGAATGCA 1101D  D  L  D  L  M  N  S  S  G  G  H  S  E  A  H 3301GATGATCTGGACCTAATGAATTCCTCAGGAGGCCATTCTGAGGCACAC 1MSKLRMVLLE DSGSADVRRH FVNLSPFTIA VVLLLRACFV TSSLGGTTKE LRLVDGENKC SEQ ID61 SGRVEVKIQE EWGTVCNNGW SMEAVSVICN QLGCPTAIKA TGWANSSAGS GRIWMDHVSCNO: 34 121RGNESALWDC KHDGWGKHSN CTHQQDAGVT CSDGSDLEMR LTNGGNMCSG RIEIKFQGQW 181GTVCDDNFNI NHASVVCKQL ECGSAVSFSG SANFGEGSGP IWFDDLICNG NESALWNCKH 241QGWGKHNCDH AEDAGVICSK GADLSLRLVD GVTECSGRLE VRFQGEWGTI CDDGWDSHDA 301AVACKQLGCP TAITAIGRVN ASEGFGHIWL DSVSCQGHEP AVWQCKHHEW GKHYCNHNED 361AGVTCSDGSG LELRLRGGGS RCAGTVEVEI QRLLGKVCDR GWGLKEADVV CRQLGCGSAL 421KTSYQVYSKI QATNMWLFLS SCNGNETSLW DCKNWQWGGL TCDHYEEAKI TCSAHREPRL 481VGGDIPCSGR VEVKHGDTWG SVCDSDFSLE AASVLCRELQ CGTVVSILGG AHFGEGNGQI 541WTEEFQCEGH ESHLSLCPVA PRPEGTCSHS RDVGVVCSRY TEIRLVNGKT PCEGRVELKT 601LNAWGSLCNS HWDIEDAHVL CQQLKCGVAL STPGGAHFGK GNGQVWRHMF HCTGTEQHMG 661DCPVTALGAS LCPSGQVASV ICSGNQSQTL SSRNSSSLGP TRPTIPEESA VACIESGQLR 721LVNGGGRCAG RVEIYHEGSW GTICDDSWDL SDAHVVCRQL GCGEAINATG SAHFGEGTGP 781IWLDEMKCNG KESRIWQCHS HGWGQQNCRH KEDAGVICSE FMSLRLTSEA SREACAGRLE 841VFYNGAWGSV GRSNMSETTV GVVCRQLGCA DKGKINSASL DKAMSIPMWV DNVQCPKGPD 901TLWQCPSSPW EKRLARPSEE TWITCDNKMR LQEGPTSCSG RVEIWHGGSW GTVCDDSWDL 961NDAQVVCQQL GCGPALKAFK EAEFGQGTGP IWLNEVKCEG NESSLWDCPA RRWGHSECGH 1021KEDAAVNCTD ISTRKTPQKA TTGQSSLIAV GILGVVLLAI FVALFLTQKR RQRQRLTVSS 1081RGENLVHQIQ YREMNSCLNA DDLDLMNSSG GHSEAH 1M  S  K  L  R  M  V  L  L  E  D  S  G  S  A  D  V  R  R  H SEQ ID 1ATGAGCAAACTCAGAATGGTGCTACTTGAAGACTCTGGATCTGCTGACGTCAGAAGACAT NO: 35 21F  V  N  L  S  P  F  T  I  A  V  V  L  L  L  R  A  C  F  V and 36 61TTTGTCAACTTGAGTCCCTTCACTATTGCTGTGGTCTTACTTCTCCGTGCCTGTTTTGTC 41T  S  S  L  G  G  T  T  K  E  L  R  L  V  D  G  E  N  K  C 121ACCAGTTCTCTTGGAGGAACAACCAAGGAGCTGAGGCTAGTGGATGGTGAAAACAAGTGT 61S  G  R  V  E  V  K  I  Q  E  E  W  G  T  V  C  N  N  G  W 181AGTGGGAGAGTGGAAGTGAAAATCCAGGAGGAGTGGGGAACGGTGTGTAATAATGGCTGG 81S  M  E  A  V  S  V  I  C  N  Q  L  G  C  P  T  A  I  K  A 241AGCATGGAAGCAGTCTCTGTGATTTGTAACCAGCTGGGATGTCCAACTGCTATCAAAGCC 101T  G  W  A  N  S  S  A  G  S  G  R  I  W  M  D  H  V  S  C 301ACTGGATGGGCTAATTCCAGTGCAGGTTCTGGACGCATTTGGATGGATCATGTTTCTTGT 121R  G  N  E  S  A  L  W  D  C  K  H  D  G  W  G  K  H  S  N 361CGTGGGAATGAGTCAGCTCTTTGGGACTGCAAACATGATGGATGGGGAAAGCATAGTAAC 141C  T  H  Q  Q  D  A  G  V  T  C  S  D  G  S  D  L  E  M  R 421TGTACTCACCAACAAGATGCTGGAGTAACTTGCTCAGATGGATCCGATTTGGAAATGAGG 161L  T  N  G  G  N  M  C  S  G  R  I  E  I  K  F  Q  G  Q  W 481CTGACGAATGGAGGGAATATGTGTTCTGGAAGAATAGAGATCAAATTCCAAGGACAGTGG 181G  T  V  C  D  D  N  F  N  I  N  H  A  S  V  V  C  K  Q  L 541GGAACAGTGTGTGATGATAACTTCAACATCAATCATGCATCTGTGGTTTGTAAACAACTT 201E  C  G  S  A  V  S  F  S  G  S  A  N  F  G  E  G  S  G  P 601GAATGTGGAAGTGCTGTCAGTTTCTCTGGTTCAGCTAATTTTGGAGAAGGCTCTGGACCA 221I  W  F  D  D  L  I  C  N  G  N  E  S  A  L  W  N  C  K  H 661ATCTGGTTTGATGATCTTATATGCAACGGAAATGAGTCAGCTCTCTGGAACTGCAAACAT 241Q  G  W  G  K  H  N  C  D  H  A  E  D  A  G  V  I  C  S  K 721CAAGGATGGGGAAAGCATAACTGTGATCATGCTGAGGATGCTGGAGTGATTTGCTCAAAG 261G  A  D  L  S  L  R  L  V  D  G  V  T  E  C  S  G  R  L  E 781GGAGCAGATCTGAGCCTGAGACTGGTAGATGGAGTCACTGAATGTTCAGGAAGATTAGAA 281V  R  F  Q  G  E  W  G  T  I  C  D  D  G  W  D  S  H  D  A 841GTGAGATTCCAAGGAGAATGGGGGACAATATGTGATGACGGCTGGGACAGTCATGATGCT 301A  V  A  C  K  Q  L  G  C  P  T  A  I  T  A  I  G  R  V  N 901GCTGTGGCATGCAAGCAACTGGGATGTCCAACTGCTATCACCGCCATTGGTCGAGTTAAC 321A  S  E  G  F  G  H  I  W  L  D  S  V  S  C  Q  G  H  E  P 961GCCAGTGAGGGATTTGGACACATCTGGCTTGACAGTGTTTCTTGCCAGGGACATGAACCT 341A  V  W  Q  C  K  H  H  E  W  G  K  H  Y  C  N  H  N  E  D 1021GCGGTCTGGCAATGTAAACACCATGAATGGGGAAAGCATTATTGCAATCACAATGAAGAT 361A  G  V  T  C  S  D  G  S  D  L  E  L  R  L  R  G  G  G  S 1081GCTGGCGTAACATGTTCTGATGGATCAGATCTGGAGCTAAGACTTAGAGGTGGAGGCAGC 381R  C  A  G  T  V  E  V  E  I  Q  R  L  L  G  K  V  C  D  R 1141CGCTGTGCTGGgACAGTTGAGGTgGAGATTCAGAGACTGTTAGGGAAGGTGTGTGACAGA 401G  W  G  L  K  E  A  D  V  V  C  R  Q  L  G  C  G  S  A  L 1201GGCTGGGGACTGAAAGAAGCTGATGTGGTTTGCAGGCAGCTGGGATGTGGATCTGCACTC 421K  T  S  Y  Q  V  Y  S  K  I  Q  A  T  N  M  W  L  F  L  S 1261AAAACATCCTATCAAGTATACTCCAAAATCCAGGCAACAAACATGTGGCTGTTTCTAAGT 441S  C  N  G  N  E  T  S  L  W  D  C  K  N  W  Q  W  G  G  L 1321AGCTGTAACGGAAATGAAACTTCTCTTTGGGACTGCAAGAACTGGCAATGGGGTGGACTT 461T  C  D  H  Y  E  E  A  K  I  T  C  S  A  H  R  E  P  R  L 1381ACCTGTGATCACTATGAAGAAGCCAAAATTACCTGCTCAGCCCACAGGGAACCCAGACTG 481V  G  G  D  I  P  C  S  G  R  V  E  V  K  H  G  D  T  W  G 1441GTTGGAGGAGACATTCCCTGTTCTGGACGCGTTGAAGTGAAGCATGGTGACACATGGGGC 501S  V  C  D  S  D  F  S  L  E  A  A  S  V  L  C  R  E  L  Q 1501TCCGTCTGTGATTCGGATTTCTCTCTGGAAGCTGCCAGCGTTCTATGCAGGGAATTACAG 521C  G  T  V  V  S  I  L  G  G  A  H  F  G  E  G  N  G  Q  I 1561TGTGGCACAGTCGTCTCTATCCTGGGGGGAGCTCACTTTGGAGAGGGAAATGGACAGATC 541W  T  E  E  F  Q  C  E  G  H  E  S  H  L  S  L  C  P  V  A 1621TGGACTGAAGAATTCCAGTGTGAGGGACATGAGTCCCATCTTTCACTCTGCCCAGTAGCA 561P  R  P  E  G  T  C  S  H  S  R  D  V  G  V  V  C  S  R  Y 1681CCCCGCCCAGAAGGAACTTGTAGCCACAGCAGGGATGTTGGAGTAGTCTGCTCAAGATAC 581T  E  I  R  L  V  N  G  K  T  P  C  E  G  R  V  E  L  K  T 1741ACAGAAATTCGCTTGGTGAATGGCAAGACCCCATGTGAGGGCAGAGTGGAGCTCAAAACG 601L  N  A  W  G  S  L  C  N  S  H  W  D  I  E  D  A  H  V  L 1801CTTAATGCCTGGGGATCCCTCTGCAACTCTCACTGGGACATAGAAGATGCCCACGTTCTT 621C  Q  Q  L  K  C  G  V  A  L  S  T  P  G  G  A  H  F  G  K 1861TGCCAACAACTTAAATGTGGAGTTGCCCTTTCTACCCCAGGAGGAGCACATTTTGGAAAA 641G  N  G  Q  V  W  R  H  M  F  H  C  T  G  T  E  Q  H  M  G 1921GGAAATGGTCAGGTCTGGAGGCATATGTTTCACTGCACTGGGACTGAGCAGCACATGGGA 661D  C  P  V  T  A  L  G  A  S  L  C  P  S  G  Q  V  A  S  V 1981GATTGTCCTGTAACTGCTCTGGGTGCTTCACTATGTCCTTCAGGGCAAGTGGCCTCTGTA 681I  C  S  G  N  Q  S  Q  T  L  S  S  C  N  S  S  S  L  G  P 2041ATTTGCTCAGGAAACCAGTCCCAAACACTGTCCTCGTGCAATTCATCATCTCTGGGCCCA 701T  R  P  T  I  P  E  E  S  A  V  A  C  I  E  S  G  Q  L  R 2101ACAAGGCCTACCATTCCAGAAGAAAGTGCTGTGGCCTGCATAGAGAGTGGTCAACTTCGC 721L  V  N  G  G  G  R  C  A  G  R  V  E  I  Y  H  E  G  S  W 2161TTGGTAAATGGAGGAGGTCGCTGTGCTGGGAGAGTAGAGATTTATCATGAGGGCTCCTGG 741G  T  I  C  D  D  S  W  D  L  S  D  A  H  V  V  C  R  Q  L 2221GGCACCATCTGTGATGACAGCTGGGACCTGAGCGATGCCCACGTGGTGTGCAGACAGCTG 761G  C  G  E  A  I  N  A  T  G  S  A  H  F  G  E  G  T  G  P 2281GGCTGTGGAGAGGCCATTAATGCCACTGGTTCTGCTCATTTTGGAGAAGGAACAGGGCCC 781I  W  L  D  E  M  K  C  N  G  K  E  S  R  I  W  Q  C  H  S 2341ATCTGGCTGGATGAGATGAAATGCAATGGAAAAGAATCCCGCATTTGGCAGTGCCATTCA 801H  G  W  G  Q  Q  N  C  R  H  K  E  D  A  G  V  I  C  S  E 2401CATGGCTGGGGGCAGCAAAACTGCAGGCACAAGGAGGATGCAGGAGTTATCTGCTCAGAG 821F  M  S  L  R  L  T  S  E  A  S  R  E  A  C  A  G  R  L  E 2461TTCATGTCTCTGAGACTGACCAGTGAAGCCAGCAGAGAGGCCTGTGCAGGGCGTCTAGAA 841V  F  Y  N  G  A  W  G  S  V  G  R  S  N  M  S  E  T  T  V 2521GTTTTTTACAACGGAGCTTGGGGCAGTGTTGGCAGGAGTAACATGTCTGAAACCACTGTG 861G  V  V  C  R  Q  L  G  C  A  D  K  G  K  I  N  S  A  S  L 2581GGTGTAGTGTGCAGGCAGCTGGGCTGTGCAGACAAAGGGAAAATCAACTCTGCATCTTTA 881D  K  A  M  S  I  P  M  W  V  D  N  V  Q  C  P  K  G  P  D 2641GACAAGGCCATGTCCATTCCCATGTGGGTGGACAATGTTCAGTGTCCAAAAGGACCTGAC 901T  L  W  Q  C  P  S  S  P  W  E  K  R  L  A  R  P  S  E  E 2701ACGCTGTGGCAGTGCCCATCATCTCCATGGGAGAAGAGACTGGCCAGGCCCTCGGAGGAG 921T  W  I  T  C  D  N  K  M  R  L  Q  E  G  P  T  S  C  S  G 2761ACCTGGATCACATGTGACAACAAGATGAGACTACAAGAAGGACCCACTTCCTGTTCTGGA 941R  V  E  I  W  H  G  G  S  W  G  T  V  C  D  D  S  W  D  L 2821CGTGTGGAGATCTGGCACGGAGGTTCCTGGGGGACAGTGTGTGATGACTCCTGGGACTTG 961N  D  A  Q  V  V  C  Q  Q  L  G  C  G  P  A  L  K  A  F  K 2881AACGATGCTCAGGTGGTGTGTCAACAACTTGGCTGTGGTCCAGCTTTGAAAGCATTCAAA 981E  A  E  F  G  Q  G  T  G  P  I  W  L  N  E  V  K  C  K  G 2941GAAGCAGAGTTTGGTCAGGGGACTGGACCCATATGGCTCAATGAAGTGAAGTGCAAAGGG 1001N  E  S  S  L  W  D  C  P  A  R  R  W  G  H  S  E  C  G  H 3001AATGAGTCTTCCTTGTGGGATTGTCCTGCCAGACGCTGGGGCCACAGTGAGTGTGGACAC 1021K  E  D  A  A  V  N  C  T  D  I  S  T  R  K  T  P  Q  K  A 3061AAGGAAGACGCTGCAGTGAATTGCACAGATATTTCAACGCGCAAAACCCCACAAAAAGCC 1041T  T  G  Q  S  S  L  I  A  V  G  I  L  G  V  V  L  L  A  I 3121ACAACAGGTCAGTCATCCCTTATTGCAGTCGGAATCCTTGGAGTTGTTCTCTTGGCCATT 1061F  V  A  L  F  L  T  Q  K  R  R  Q  R  Q  R  L  T  V  S  S 3181TTCGTCGCATTATTCTTGACTCAAAAGCGAAGACAGAGACAGCGGCTTACAGTTTCCTCA 1081R  G  E  N  L  V  H  Q  I  Q  Y  R  E  M  N  S  C  L  N  A 3241AGAGGAGAGAACTTAGTCCACCAAATTCAATACCGGGAGATGAATTCTTGCCTGAATGCA 1101D  D  L  D  L  M  N  S  S  E  N  S  N  E  S  A  D  F  N  A 3301GATGATCTGGACCTAATGAATTCCTCAGAAAATTCCAATGAGTCAGCTGATTTCAATGCT 1121A  E  L  I  S  V  S  K  F  L  P  I  S  G  M  E  K  E  A  I 3361GCTGAACTAATTTCTGTGTCTAAATTTCTTCCTATTTCTGGAATGGAAAAGGAGGCCATT 1141L  R  H  T  E  K  E  N  G  N  L 3421 CTGAGGCACACTGAAAAGGAAAATGGGAATTTA 1MSKLRMVLLE DSGSADVRRH FVNLSPFTIA VVLLLRACFV TSSLGGTTKE LRLVDGENKC SEQ ID61 SGRVEVKIQE EWGTVCNNGW SMEAVSVICN QLGCPTAIKA TGWANSSAGS GRIWMDHVSCNO: 36 121RGNESALWDC KHDGWGKHSN CTHQQDAGVT CSDGSDLEMR LTNGGNMCSG RIEIKFQGQW 181GTVCDDNFNI NHASVVCKQL ECGSAVSFSG SANFGEGSGP IWFDDLICNG NESALWNCKH 241QGWGKHNCDH AEDAGVICSK GADLSLRLVD GVTECSGRLE VRFQGEWGTI CDDGWDSHDA 301AVACKQLGCP TAITAIGRVN ASEGFGHIWL DSVSCQGHEP AVWQCKHHEW GKHYCNHNED 361AGVTCSDGSD LELRLRGGGS RCAGTVEVEI QRLLGKVCDR GWGLKEADVV CRQLGCGSAL 421KTSYQVYSKI QATNMWLFLS SCNGNETSLW DCKNWQWGGL TCDHYEEAKI TCSAHREPRL 481VGGDIPCSGR VEVKHGDTWG SVCDSDFSLE AASVLCRELQ CGTVVSILGG AHFGEGNGQI 541WTEEFQCEGH ESHLSLCPVA PRPEGTCSHS RDVGVVCSRY TEIRLVNGKT PCEGRVELKT 601LNAWGSLCNS HWDIEDAHVL CQQLKCGVAL STPGGAHFGK GNGQVWRHMF HCTGTEQHMG 661DCPVTALGAS LCPSGQVASV ICSGNQSQTL SSCNSSSLGP TRPTIPEESA VACIESGQLR 721LVNGGGRCAG RVEIYHEGSW GTICDDSWDL SDAHVVCRQL GCGEAINATG SAHFGEGTGP 781IWLDEMKCNG KESRIWQCHS HGWGQQNCRH KEDAGVICSE FMSLRLTSEA SREACAGRLE 841VFYNGAWGSV GRSNMSETTV GVVCRQLGCA DKGKINSASL DKAMSIPMWV DNVQCPKGPD 901TLWQCPSSPW EKRLARPSEE TWITCDNKMR LQEGPTSCSG RVEIWHGGSW GTVCDDSWDL 961NDAQVVCQQL GCGPALKAFK EAEFGQGTGP IWLNEVKCKG NESSLWDCPA RRWGHSECGH 1021KEDAAVNCTD ISTRKTPQKA TTGQSSLIAV GILGVVLLAI FVALFLTQKR RQRQRLTVSS 1081RGENLVHQIQ YREMNSCLNA DDLDLMNSSE NSNESADFNA AELISVSKFL PISGMEKEAI 1141LRHTEKENGN L 1M  S  K  L  R  M  V  L  L  E  D  S  G  S  A  D  V  R  R  H SEQ ID 1ATGAGCAAACTCAGAATGGTGCTACTTGAAGACTCTGGATCTGCTGACGTCAGAAGACAT NO: 37 21F  V  N  L  S  P  F  T  I  A  V  V  L  L  L  R  A  C  F  V and 38 61TTTGTCAACTTGAGTCCCTTCACTATTGCTGTGGTCTTACTTCTCCGTGCCTGTTTTGTC 41T  S  S  L  G  G  T  T  K  E  L  R  L  V  D  G  E  N  K  C 121ACCAGTTCTCTTGGAGGAACAACCAAGGAGCTGAGGCTAGTGGATGGTGAAAACAAGTGT 61S  G  R  V  E  V  K  I  Q  E  E  W  G  T  V  C  N  N  G  W 181AGTGGGAGAGTGGAAGTGAAAATCCAGGAGGAGTGGGGAACGGTGTGTAATAATGGCTGG 81S  M  E  A  V  S  V  I  C  N  Q  L  G  C  P  T  A  I  K  A 241AGCATGGAAGCAGTCTCTGTGATTTGTAACCAGCTGGGATGTCCAACTGCTATCAAAGCC 101T  G  W  A  N  S  S  A  G  S  G  R  I  W  M  D  H  V  S  C 301ACTGGATGGGCTAATTCCAGTGCAGGTTCTGGACGCATTTGGATGGATCATGTTTCTTGT 121R  G  N  E  S  A  L  W  D  C  K  H  D  G  W  G  K  H  S  N 361CGTGGGAATGAGTCAGCTCTTTGGGACTGCAAACATGATGGATGGGGAAAGCATAGTAAC 141C  T  H  Q  Q  D  A  G  V  T  C  S  D  G  S  D  L  E  M  R 421TGTACTCACCAACAAGATGCTGGAGTAACTTGCTCAGATGGATCCGATTTGGAAATGAGG 161L  T  N  G  G  N  M  C  S  G  R  I  E  I  K  F  Q  G  Q  W 481CTGACGAATGGAGGGAATATGTGTTCTGGAAGAATAGAGATCAAATTCCAAGGACAGTGG 181G  T  V  C  D  D  N  F  N  I  N  H  A  S  V  V  C  K  Q  L 541GGAACAGTGTGTGATGATAACTTCAACATCAATCATGCATCTGTGGTTTGTAAACAACTT 201E  C  G  S  A  V  S  F  S  G  S  A  N  F  G  E  G  S  G  P 601GAATGTGGAAGTGCTGTCAGTTTCTCTGGTTCAGCTAATTTTGGAGAAGGCTCTGGACCA 221I  W  F  D  D  L  I  C  N  G  N  E  S  A  L  W  N  C  K  H 661ATCTGGTTTGATGATCTTATATGCAACGGAAATGAGICAGCTCTCTGGAACTGCAAACAT 241Q  G  W  G  K  H  N  C  D  H  A  E  D  A  G  V  I  C  S  K 721CAAGGATGGGGAAAGCATAACTGTGATCATGCTGAGGATGCTGGAGTGATTTGCTCAAAG 261G  A  D  L  S  L  R  L  V  D  G  V  T  E  C  S  G  R  L  E 781GGAGCAGATCTGAGCCTGAGACTGGTAGATGGAGTCACTGAATGTTCAGGAAGATTAGAA 281V  R  F  Q  G  E  W  G  T  I  C  D  D  G  W  D  S  H  D  A 841GTGAGATTCCAAGGAGAATGGGGGACAATATGTGATGACGGCTGGGACAGTCATGATGCT 301A  V  A  Q  L  G  C  P  T  A  I  T  A  I  G  R  V  N 901GCTGTGGCATGCAAGCAACTGGGATGTCCAACTGCTATCACCGCCATTGGTCGAGTTAAC 321A  S  E  G  F  G  H  I  W  L  D  S  V  S  C  Q  G  H  E  P 961GCCAGTGAGGGATTTGGACACATCTGGCTTGACAGTGTTTCTTGCCAGGGACATGAACCT 341A  V  W  Q  C  K  H  H  E  W  G  K  H  Y  C  N  H  N  E  D 1021GCGGTCTGGCAATGTAAACACCATGAATGGGGAAAGCATTATTGCAATCACAATGAAGAT 361A  G  V  T  C  S  D  G  S  D  L  E  L  R  L  R  G  G  G  S 1081GCTGGCGTAACATGTTCTGATGGATCAGATCTGGAGCTAAGACTTAGAGGTGGAGGCAGC 381R  C  A  G  T  V  E  V  E  I  Q  R  L  L  G  K  V  C  D  R 1141CGCTGTGCTGGGACAGTTGAGGTGGAGATTCAGAGACTGTTAGGGAAGGTGTGTGACAGA 401G  W  G  L  K  E  A  D  V  V  C  R  Q  L  G  C  G  S  A  L 1201GGCTGGGGACTGAAAGAAGCTGATGTGGTTTGCAGGCAGCTGGGATGTGGATCTGCACTC 421K  T  S  Y  Q  V  Y  S  K  I  Q  A  T  N  M  W  L  F  L  S 1261AAAACATCCTATCAAGTATACTCCAAAATCCAGGCAACAAACATGTGGCTGTTTCTAAGT 441S  C  N  G  N  E  T  S  L  W  D  C  K  N  W  Q  W  G  G  L 1321AGCTGTAACGGAAATGAAACTTCTCTTTGGGACTGCAAGAACTGGCAATGGGGTGGACTT 461T  C  D  H  Y  E  E  A  K  I  T  C  S  A  H  R  E  P  R  L 1381ACCTGTGATCACTATGAAGAAGCCAAAATTACCTGCTCAGCCCACAGGGAACCCAGACTG 481V  G  G  D  I  P  C  S  G  R  V  E  V  K  H  G  D  T  W  G 1441GTTGGAGGAGACATTCCCTGTTCTGGACGCGTTGAAGTGAAGCATGGTGACACATGGGGC 501S  V  C  D  S  D  F  S  L  E  A  A  S  V  L  C  R  E  L  Q 1501TCCGTCTGTGATTCGGATTTCTCTCTGGAAGCTGCCAGCGTTCTATGCAGGGAATTACAG 521C  G  T  V  V  S  I  L  G  G  A  H  F  G  E  G  N  G  Q  I 1561TGTGGCACAGTCGTCTCTATCCTGGGGGGAGCTCACTTTGGAGAGGGAAATGGACAGATC 541W  T  E  E  F  Q  C  E  G  H  E  S  H  L  S  L  C  P  V  A 1621TGGACTGAAGAATTCCAGTGTGAGGGACATGAGTCCCATCTTTCACTCTGCCCAGTAGCA 561P  R  P  E  G  T  C  S  H  S  R  D  V  G  V  V  C  S  R  Y 1681CCCCGCCCAGAAGGAACTTGTAGCCACAGCAGGGATGTTGGAGTAGTCTGCTCAAGATAC 581T  E  I  R  L  V  N  G  K  T  P  C  E  G  R  V  E  L  K  T 1741ACAGAAATTCGCTTGGTGAATGGCAAGACCCCATGTGAGGGCAGAGTGGAGCTCAAAACG 601L  N  A  W  G  S  L  C  N  S  H  W  D  I  E  D  A  H  V  L 1801CTTAATGCCTGGGGATCCCTCTGCAACTCTCACTGGGACATAGAAGATGCCCACGTTCTT 621C  Q  Q  L  K  C  G  V  A  L  S  T  P  G  G  A  H  F  G  K 1861TGCCAACAACTTAAATGTGGAGTTGCCCTTTCTACCCCAGGAGGAGCACATTTTGGAAAA 641G  N  G  Q  V  W  R  H  M  F  H  C  T  G  T  E  Q  H  M  G 1921GGAAATGGTCAGGTCTGGAGGCATATGTTTCACTGCACTGGGACTGAGCAGCACATGGGA 661D  C  P  V  T  A  L  G  A  S  L  C  P  S  G  Q  V  A  S  V 1981GATTGTCCTGTAACTGCTCTGGGTGCTTCACTATGTCCTTCAGGGCAAGTGGCCTCTGTA 681I  C  S  G  N  Q  S  Q  T  L  S  S  C  N  S  S  S  L  G  P 2041ATTTGCTCAGGAAACCAGTCCCAAACACTGTCCTCGTGCAATTCATCATCTCTGGGCCCA 701T  R  P  T  I  P  E  E  S  A  V  A  C  I  E  S  G  Q  L  R 2101ACAAGGCCTACCATTCCAGAAGAAAGTGCTGTGGCCTGCATAGAGAGTGGTCAACTTCGC 721L  V  N  G  G  G  R  C  A  G  R  V  E  I  Y  H  E  G  S  W 2161TTGGTAAATGGAGGAGGTCGCTGTGCTGGGAGAGTAGAGATTTATCATGAGGGCTCCTGG 741G  T  I  C  D  D  S  W  D  L  S  D  A  H  V  V  C  R  Q  L 2221GGCACCATCTGTGATGACAGCTGGGACCTGAGCGATGCCCACGTGGTGTGCAGACAGCTG 761G  C  G  E  A  I  N  A  T  G  S  A  H  F  G  E  G  T  G  P 2281GGCTGTGGAGAGGCCATTAATGCCACTGGTTCTGCTCATTTTGGAGAAGGAACAGGGCCC 781I  W  L  D  E  M  K  C  N  G  K  E  S  R  I  W  Q  C  H  S 2341ATCTGGCTGGATGAGATGAAATGCAATGGAAAAGAATCCCGCATTTGGCAGTGCCATTCA 801H  G  W  G  Q  Q  N  C  R  H  K  E  D  A  G  V  I  C  S  E 2401CATGGCTGGGGGCAGCAAAACTGCAGGCACAAGGAGGATGCAGGAGTTATCTGCTCAGAG 821F  M  S  L  R  L  T  S  E  A  S  R  E  A  C  A  G  R  L  E 2461TTCATGTCTCTGAGACTGACCAGTGAAGCCAGCAGAGAGGCCTGTGCAGGGCGTCTAGAA 841V  F  Y  N  G  A  W  G  S  V  G  R  S  N  M  S  E  T  T  V 2521GTTTTTTACAACGGAGCTTGGGGCAGTGTTGGCAGGAGTAACATGTCTGAAACCACTGTG 861G  V  V  C  R  Q  L  G  C  A  D  K  G  K  I  N  S  A  S  L 2581GGTGTAGTGTGCAGGCAGCTGGGCTGTGCAGACAAAGGGAAAATCAACTCTGCATCTTTA 881D  K  A  M  S  I  P  M  W  V  D  N  V  Q  C  P  K  G  P  D 2641GACAAGGCCATGTCCATTCCCATGTGGGTGGACAATGTTCAGTGTCCAAAAGGACCTGAC 901T  L  W  Q  C  P  S  S  P  W  E  K  R  L  A  R  P  S  E  E 2701ACGCTGTGGCAGTGCCCATCATCTCCATGGGAGAAGAGACTGGCCAGGCCCTCGGAGGAG 921T  W  I  T  C  D  N  K  M  R  L  Q  E  G  P  T  S  C  S  G 2761ACCTGGATCACATGTGACAACAAGATGAGACTACAAGAAGGACCCACTTCCTGTTCTGGA 941R  V  E  I  W  H  G  G  S  W  G  T  V  C  D  D  S  W  D  L 2821CGTGTGGAGATCTGGCACGGAGGTTCCTGGGGGACAGTGTGTGATGACTCCTGGGACTTG 961N  D  A  Q  V  V  C  Q  Q  L  G  C  G  P  A  L  K  A  F  K 2881AACGATGCTCAGGTGGTGTGTCAACAACTTGGCTGTGGTCCAGCTTTGAAAGCATTCAAA 981E  A  E  F  G  Q  G  T  G  P  I  W  L  N  E  V  K  C  K  G 2941GAAGCAGAGTTTGGTCAGGGGACTGGACCCATATGGCTCAATGAAGTGAAGTGCAAAGGG 1001N  E  S  S  L  W  D  C  P  A  R  R  W  G  H  S  E  C  G  H 3001AATGAGTCTTCCTTGTGGGATTGTCCTGCCAGACGCTGGGGCCACAGTGAGTGTGGACAC 1021K  E  D  A  A  V  N  C  T  D  I  S  T  R  K  T  P  Q  K  A 3061AAGGAAGACGCTGCAGTGAATTGCACAGATATTTCAACGCGCAAAACCCCACAAAAAGCC 1041T  T  V  S  S  R  G  E  N  L  V  H  Q  I  Q  Y  R  E  M  N 3121ACAACGGTTTCCTCAAGAGGAGAGAACTTAGTCCACCAAATTCAATACCGGGAGATGAAT 1061S  C  L  N  A  D  D  L  N  L  M  N  S  S  G  G  H  S  E  A 3181TCTTGCCTGAATGCAGATGATCTGAACCTAATGAATTCCTCAGGAGGCCATTCTGAGGCA 1081H  *  K  G  K  W  E  F  I  T  Q 3241 CACTGAAAAGGAAAATGGGAATTTATAACCCAG 1MSKLRMVLLE DSGSADVRRH FVNLSPFTIA VVLLLRACFV TSSLGGTTKE LRLVDGENKC SEQ ID61 SGRVEVKIQE EWGTVCNNGW SMEAVSVICN QLGCPTAIKA TGWANSSAGS GRIWMDHVSCNO: 38 121RGNESALWDC KHDGWGKHSN CTHQQDAGVT CSDGSDLEMR LTNGGNMCSG RIEIKFQGQW 181GTVCDDNFNI NHASVVCKQL ECGSAVSFSG SANFGEGSGP IWFDDLICNG NESALWNCKH 241QGWGKHNCDH AEDAGVICSK GADLSLRLVD GVTECSGRLE VRFQGEWGTI CDDGWDSHDA 301AVACKQLGCP TAITAIGRVN ASEGFGHIWL DSVSCQGHEP AVWQCKHHEW GKHYCNHNED 361AGVTCSDGSD LELRLRGGGS RCAGTVEVEI QRLLGKVCDR GWGLKEADVV CRQLGCGSAL 421KTSYQVYSKI QATNMWLFLS SCNGNETSLW DCKNWQWGGL TCDHYEEAKI TCSAHREPRL 481VGGDIPCSGR VEVKHGDTWG SVCDSDFSLE AASVLCRELQ CGTVVSILGG AHFGEGNGQI 541WTEEFQCEGH ESHLSLCPVA PRPEGTCSHS RDVGVVCSRY TEIRLVNGKT PCEGRVELKT 601LNAWGSLCNS HWDIEDAHVL CQQLKCGVAL STPGGAHFGK GNGQVWRHMF HCTGTEQHMG 661DCPVTALGAS LCPSGQVASV ICSGNQSQTL SSCNSSSLGP TRPTIPEESA VACIESGQLR 721LVNGGGRCAG RVEIYHEGSW GTICDDSWDL SDAHVVCRQL GCGEAINATG SAHFGEGTGP 781IWLDEMKCNG KESRIWQCHS HGWGQQNCRH KEDAGVICSE FMSLRLTSEA SREACAGRLE 841VFYNGAWGSV GRSNMSETTV GVVCRQLGCA DKGKINSASL DKAMSIPMWV DNVQCPKGPD 901TLWQCPSSPW EKRLARPSEE TWITCDNKMR LQEGPTSCSG RVEIWHGGSW GTVCDDSWDL 961NDAQVVCQQL GCGPALKAFK EAEFGQGTGP IWLNEVKCKG NESSLWDCPA RRWGHSECGH 1021KEDAAVNCTD ISTRKTPQKA TTVSSRGENL VHQIQYREMN SCLNADDLNL MNSSGGHSEA 1081 H1 M  S  K  L  R  M  V  L  L  E  D  S  G  S  A  D  V  R  R  H SEQ ID 1ATGAGCAAACTCaGAATGGTGCTACTTGAAGACTCTGGATCTGCTGACGTCAGAAGACAT NO: 39 21F  V  N  L  S  P  F  T  I  A  V  V  L  L  L  R  A  C  F  V and 40 61TTTGTCAACTTGAGTCCCTTCACTATTGCTGTGGTCTTACTTCTCCGTGCCTGTTTTGTC 41T  S  S  L  G  G  T  T  K  E  L  R  L  V  D  G  E  N  K  C 121ACCAGTTCTCTTGGAGGAACAACCAAGGAGCTGAGGCTAGTGGATGGTGAAAACAAGTGT 61S  G  R  V  E  V  K  I  Q  E  E  W  G  T  V  C  N  N  G  W 181AGTGGGAGAGTGGAAGTGAAAATCCAGGAGGAGTGGGGAACGGTGTGTAATAATGGCTGG 81S  M  E  A  V  S  V  I  C  N  Q  L  G  C  P  T  A  I  K  A 241AGCATGGAAGCAGTCTCTGTGATTTGTAACCAGCTGGGATGTCCAACTGCTATCAAAGCC 101T  G  W  A  N  S  S  A  G  S  G  R  I  W  M  D  H  V  S  C 301ACTGGATGGGCTAATTCCAGTGCAGGTTCTGGACGCATTTGGATGGATCATGTTTCTTGT 121R  G  N  E  S  A  L  W  D  C  K  H  D  G  W  G  K  H  S  N 361CGTGGGAATGAGTCAGCTCTTTGGGACTGCAAACATGATGGATGGGGAAAGCATAGTAAC 141C  T  H  Q  Q  D  A  G  V  T  C  S  D  G  S  D  L  E  M  R 421TGTACTCACCAACAAGATGCTGGAGTGACTTGCTCAGATGGATCCGATTTGGAAATGAGG 161L  T  N  G  G  N  M  C  S  G  R  I  E  I  K  F  Q  G  Q  W 481CTGACGAATGGAGGGAATATGTGTTCTGGAAGAATAGAGATCAAATTCCAAGGACAGTGG 181G  T  V  C  D  D  N  F  N  V  N  H  A  S  V  V  C  K  Q  L 541GGAACAGTGTGTGATGATAACTTCAACGTCAATCATGCATCTGTGGTTTGTAAACAACTT 201E  C  G  S  A  V  S  F  S  G  S  A  N  F  G  E  G  S  G  P 601GAATGTGGAAGTGCTGTCAGTTTCTCTGGTTCAGCTAATTTTGGAGAAGGCTCTGGACCA 221I  W  F  D  D  L  I  C  N  G  N  E  S  A  L  W  N  C  K  H 661ATCTGGTTTGATGATCTTATATGCAACGGAAATGAGTCAGCTCTCTGGAACTGCAAACAT 241Q  G  W  G  K  H  N  C  D  H  A  E  D  A  G  V  I  C  S  K 721CAAGGATGGGGAAAGCATAACTGTGATCATGCTGAGGATGCTGGAGTGATTTGCTCAAAG 261G  A  D  L  S  L  R  L  V  D  G  V  T  E  C  S  G  R  L  E 781GGAGCAGATCTGAGCCTGAGACTGGTAGATGGAGTCACTGAATGTTCAGGAAGATTAGAA 281V  R  F  Q  G  E  W  G  T  I  C  D  D  G  W  D  S  H  D  A 841GTGAGATTCCAAGGAGAATGGGGGACAATATGTGATGACGGCTGGGACAGTCATGATGCT 301A  V  A  C  K  Q  L  G  C  P  T  A  I  T  A  I  G  R  V  N 901GCTGTGGCATGCAAGCAACTGGGATGTCCAACTGCTATCACCGCCATTGGTCGAGTTAAC 321A  S  E  G  F  G  H  I  W  L  D  S  V  S  C  Q  G  H  E  P 961GCCAGTGAGGGATTTGGACACATCTGGCTTGACAGTGTTTCTTGCCAGGGACATGAACCT 341A  V  W  Q  C  K  H  H  E  W  G  K  H  Y  C  N  H  N  E  D 1021GCGGTCTGGCAATGTAAACACCATGAATGGGGAAAGCATTATTGCAATCACAATGAAGaT 361A  G  V  T  C  S  D  G  S  D  L  E  L  R  L  R  G  G  G  S 1081GCTGGCGTAACATGTTCTGATGGATCAGATCTGGAGCTAAGACTTAGAGGTGGAGGCAGC 381R  C  A  G  T  V  E  V  E  I  Q  R  L  L  G  K  V  C  D  R 1141CGCTGTGCTGGGACAGTTGAGGTGGAGATtCAGAGACTGTTAGGGAAGGTGTGTGACAGA 401G  W  G  L  K  E  A  D  V  V  C  R  Q  L  G  C  G  S  A  L 1201GGCTGGGGACTGAAAGAAGCTGATGTGGTTTGCAGGCAGCTGGGATGTGGATCTGCACTC 421K  T  S  Y  Q  V  Y  S  K  I  Q  A  T  N  M  W  L  F  L  S 1261AAAACATCCTATCAAGTATACTCCAAAATCCAGGCAACAAACATGTGGCTGTTTCTAAGT 441S  C  N  G  N  E  T  S  L  W  D  C  K  N  W  Q  W  G  G  L 1321AGCTGTAACGGAAATGAAACTTCTCTTTGGGACTGCAAGAACTGGCAATGGGGTGGACTT 461T  C  D  H  Y  E  E  A  K  I  T  C  S  A  H  R  E  P  R  L 1381ACCTGTGATCACTATGAAGAAGCCAAAATTACCTGCTCAGCCCACAGGGAACCCAGACTG 481V  G  G  D  I  P  C  S  G  R  V  E  V  K  H  G  D  T  W  G 1441GTTGGAGGAGACATTCCCTGTTCTGGACGCGTTGAAGTGAAGCATGGTGACACATGGGGC 501S  V  C  D  S  D  F  S  L  E  A  A  S  V  L  C  R  E  L  Q 1501TCCGTCTGTGATTCGGATTTCTCTCTGGAAGCTGCCAGCGTTCTATGCAGGGAATTACAG 521C  G  T  V  V  S  I  L  G  G  A  H  F  G  E  G  N  G  Q  I 1561TGTGGCACAGTCGTCTCTATCCTGGGGGGAGCTCACTTTGGAGAGGGAAATGGACAGATC 541W  A  E  E  F  Q  C  E  G  H  E  S  H  L  S  L  C  P  V  A 1621TGGGCTGAAGAATTCCAGTGTGAGGGACATGAGTCCCATCTTTCACTCTGCCCAGTAGCA 561P  R  P  E  G  T  C  S  H  S  R  D  V  G  V  V  C  S  R  Y 1681CCCCGCCCAGAAGGAACTTGTAGCCACAGCAGGGATGTTGGAGTAGTCTGCTCAAGATAC 581T  E  I  R  L  V  N  G  K  T  P  C  E  G  R  V  E  L  K  T 1741ACAGAAATTCGCTTGGTGAATGGCAAGACCCCATGTGAGGGCAGAGTGGAGCTCAAAACG 601L  N  A  W  G  S  L  C  N  S  H  W  D  I  E  D  A  H  V  L 1801CTTAATGCCTGGGGATCCCTCTGCAACTCTCACTGGGACATAGAAGATGCCCACGTTCTT 621C  Q  Q  L  K  C  G  V  A  L  S  T  P  G  G  A  H  F  G  K 1861TGCCAACAACTTAAATGTGGAGTTGCCCTTTCTACCCCAGGAGGAGCACATTTTGGAAAA 641G  N  G  Q  V  W  R  H  M  F  H  C  T  G  T  E  Q  H  M  G 1921GGAAATGGTCAGGTCTGGAGGCATATGTTTCACTGCACTGGGACTGAGCAGCACATGGGA 661D  C  P  V  T  A  L  G  A  S  L  C  P  S  G  Q  V  A  S  V 1981GATTGTCCTGTAACTGCTCTGGGTGCTTCACTATGTCCTTCAGGGCAAGTGGCCTCTGTA 681I  C  S  G  N  Q  S  Q  T  L  S  S  C  N  S  S  S  L  G  P 2041ATTTGCTCAGGAAACCAGTCCCAAACACTGTCCTCGTGCAATTCATCATCTCTGGGCCCA 701T  R  P  T  I  P  E  E  S  A  V  A  C  I  E  S  G  Q  L  R 2101ACAAGGCCTACCATTCCAGAAGAAAGTGCTGTGGCCTGCATAGAGAGTGGTCAACTTCGC 721L  V  N  G  G  G  R  C  A  G  R  V  E  I  Y  H  E  G  S  W 2161TTGGTAAATGGAGGAGGTCGCTGTGCTGGGAGAGTAGAGATTTATCATGAGGGCTCCTGG 741G  T  I  C  D  D  S  W  D  L  S  D  A  H  V  V  C  R  Q  L 2221GGCACCATCTGTGATGACAGCTGGGACCTGAGCGATGCCCACGTGGTGTGCAGACAGCTG 761G  C  G  E  A  I  N  A  T  G  S  A  H  F  G  E  G  T  G  P 2281GGCTGTGGAGAGGCCATTAATGCCACTGGTTCTGCTCATTTTGGAGAAGGAACAGGGCCC 781I  W  L  D  E  M  K  C  N  G  K  E  S  R  I  W  Q  C  H  S 2341ATCTGGCTGGATGAGATGAAATGCAATGGAAAAGAATCCCGTATTTGGCAGTGCCATTCA 801H  G  W  G  Q  Q  N  C  R  H  K  E  D  A  G  V  I  C  S  E 2401CATGGCTGGGGGCAGCAAAACTGCAGGCACAAGGAGGATGCAGGAGTTATCTGCTCAGAG 821F  M  S  L  R  L  T  S  E  A  S  R  E  A  C  A  G  R  L  E 2461TTCATGTCTCTGAGACTGACCAGTGAAGCCAGCAGAGAGGCCTGTGCAGGGCGTCTAGAA 841V  F  Y  N  G  A  W  G  S  V  G  R  S  N  M  S  E  T  T  V 2521GTTTTTTACAACGGAGCTTGGGGCAGTGTTGGCAGGAGTAACATGTCTGAAACCACTGTG 861G  V  V  C  R  Q  L  G  C  A  D  K  G  K  I  N  S  A  S  L 2581GGTGTGGTGTGCAGGCAGCTGGGCTGTGCAGACAAAGGGAAAATCAACTCTGCATCTTTA 881D  K  A  M  S  I  P  M  W  V  D  N  V  Q  C  P  K  G  P  D 2641GACAAGGCCATGTCCATTCCCATGTGGGTGGACAATGTTCAGTGTCCAAAAGGACCTGAC 901T  L  W  Q  C  P  S  S  P  W  E  K  R  L  A  R  P  S  E  E 2701ACGCTGTGGCAGTGCCCATCATCTCCATGGGAGAAGAGACTGGCCAGGCCCTCGGAGGAG 921T  W  I  T  C  D  N  K  M  R  L  Q  E  G  P  T  S  C  S  G 2761ACCTGGATCACATGTGACAACAAGATGAGACTACAAGAAGGACCCACTTCCTGTTCTGGA 941R  V  E  I  W  H  G  G  S  W  G  T  V  C  D  D  S  W  D  L 2821CGTGTGGAGATCTGGCACGGAGGTTCCTGGGGGACAGTGTGTGATGACTCCTGGGACTTG 961N  D  A  Q  V  V  C  Q  Q  L  G  C  G  P  A  L  K  A  F  K 2881AACGATGCTCAGGTGGTGTGTCAACAACTTGGCTGTGGTCCAGCTTTGAAAGCATTCAAA 981E  A  E  F  G  Q  G  T  G  P  I  W  L  N  E  V  K  C  K  G 2941GAAGCAGAGTTTGGTCAGGGGACTGGACCCATATGGCTCAATGAAGTGAAGTGCAAAGGG 1001N  E  S  S  L  W  D  C  P  A  R  R  W  G  H  S  E  C  G  H 3001AATGAGTCTTCCTTGTGGGATTGTCCTGCCAGACGCTGGGGCCACAGTGAGTGTGGACAC 1021K  E  D  A  A  V  N  C  T  A  Q  K  I  S  T  H  K  T  P  Q 3061AAGGAAGACGCTGCAGTGAATTGCACAGCACAAAAAATTTCAACGCACAAAACCCCACAA 1041K  A  T  T  V  S  S  R  G  E  N  L  V  H  Q  I  Q  Y  R  E 3121AAAGCCACAACAGTTTCCTCAAGAGGAGAGAACTTAGTCCACCAAATTCAATACCGGGAG 1061M  N  S  C  L  N  A  D  D  L  D  L  M  N  S  S  G  G  H  S 3181ATGAATTCTTGCCTGAATGCAGATGATCTGGACCTAATGAATTCCTCAGGAGGCCATTCT 1081E  A  H  *  K  G  K  W  E  F  I  T  Q 3241GAGGCACACTGAAAAGGAAAATGGGAATTTATAACCCAG 1MSKLRMVLLE DSGSADVRRH FVNLSPFTIA VVLLLRACFV TSSLGGTTKE LRLVDGENKC SEQ ID61 SGRVEVKIQE EWGTVCNNGW SMEAVSVICN QLGCPTAIKA TGWANSSAGS GRIWMDHVSCNO: 40 121RGNESALWDC KHDGWGKHSN CTHQQDAGVT CSDGSDLEMR LTNGGNMCSG RIEIKFQGQW 181GTVCDDNFNV NHASVVCKQL ECGSAVSFSG SANFGEGSGP IWFDDLICNG NESALWNCKH 241QGWGKHNCDH AEDAGVICSK GADLSLRLVD GVTECSGRLE VRFQGEWGTI CDDGWDSHDA 301AVACKQLGCP TAITAIGRVN ASEGFGHIWL DSVSCQGHEP AVWQCKHHEW GKHYCNHNED 361AGVTCSDGSD LELRLRGGGS RCAGTVEVEI QRLLGKVCDR GWGLKEADVV CRQLGCGSAL 421KTSYQVYSKI QATNMWLFLS SCNGNETSLW DCKNWQWGGL TCDHYEEAKI TCSAHREPRL 481VGGDIPCSGR VEVKHGDTWG SVCDSDFSLE AASVLCRELQ CGTVVSILGG AHFGEGNGQI 541WAEEFQCEGH ESHLSLCPVA PRPEGTCSHS RDVGVVCSRY TEIRLVNGKT PCEGRVELKT 601LNAWGSLCNS HWDIEDAHVL CQQLKCGVAL STPGGAHFGK GNGQVWRHMF HCTGTEQHMG 661DCPVTALGAS LCPSGQVASV ICSGNQSQTL SSCNSSSLGP TRPTIPEESA VACIESGQLR 721LVNGGGRCAG RVEIYHEGSW GTICDDSWDL SDAHVVCRQL GCGEAINATG SAHFGEGTGP 781IWLDEMKCNG KESRIWQCHS HGWGQQNCRH KEDAGVICSE FMSLRLTSEA SREACAGRLE 841VFYNGAWGSV GRSNMSETTV GVVCRQLGCA DKGKINSASL DKAMSIPMWV DNVQCPKGPD 901TLWQCPSSPW EKRLARPSEE TWITCDNKMR LQEGPTSCSG RVEIWHGGSW GTVCDDSWDL 961NDAQVVCQQL GCGPALKAFK EAEFGQGTGP IWLNEVKCKG NESSLWDCPA RRWGHSECGH 1021KEDAAVNCTA QKISTHKTPQ KATTVSSRGE NLVHQIQYRE MNSCLNADDL DLMNSSGGHS 1081EAH 1 M  S  K  L  R  M  V  L  L  E  D  S  G  S  A  D  V  R  R  H SEQ ID1 ATGAGCAAACTCAGAATGGTGCTACTTGAAGACTCTGGATCTGCTGACGTCAGAAGACAT NO: 41 21F  V  N  L  S  P  F  T  I  A  V  V  L  L  L  R  A  C  F  V and 42 61TTTGTCAACTTGAGTCCCTTCACTATTGCTGTGGTCTTACTTCTCCGTGCCTGTTTTGTC 41T  S  S  L  G  G  T  T  K  E  L  R  L  V  D  G  E  N  K  C 121ACCAGTTCTCTTGGAGGAACAACCAAGGAGCTGAGGCTAGTGGATGGTGAAAACAAGTGT 61S  G  R  V  E  V  K  I  Q  E  E  W  G  T  V  C  N  N  G  W 181AGTGGGAGAGTGGAAGTGAAAATCCAGGAGGAGTGGGGAACGGTGTGTAATAATGGCTGG 81S  M  E  A  V  S  V  I  C  N  Q  L  G  C  P  T  A  I  K  A 241AGCATGGAAGCAGTCTCTGTGATTTGTAACCAGCTGGGATGTCCAACTGCTATCAAAGCC 101T  G  W  A  N  S  S  A  G  S  G  R  I  W  M  D  H  V  S  C 301ACTGGATGGGCTAATTCCAGTGCAGGTTCTGGACGCATTTGGATGGATCATGTTTCTTGT 121R  G  N  E  S  A  L  W  D  C  K  H  D  G  W  G  K  H  S  N 361CGTGGGAATGAGTCAGCTCTTTGGGACTGCAAACATGATGGATGGGGAAAGCATAGTAAC 141C  T  H  Q  Q  D  A  G  V  T  C  S  D  G  S  D  L  E  M  R 421TGTACTCACCAACAAGATGCTGGAGTGACTTGCTCAGATGGATCCGATTTGGAAATGAGG 161L  T  N  G  G  N  M  C  S  G  R  I  E  I  K  F  Q  G  Q  W 481CTGACGAATGGAGGGAATATGTGTTCTGGAAGAATAGAGATCAAATTCCAAGGACAGTGG 181G  T  V  C  D  D  N  F  N  I  N  H  A  S  V  V  C  K  Q  L 541GGAACAGTGTGTGATGATAACTTCAACATCAATCATGCATCTGTGGTTTGTAAACAACTT 201E  C  G  S  A  V  S  F  S  G  S  A  N  F  G  E  G  S  G  P 601GAATGTGGAAGTGCTGTCAGTTTCTCTGGTTCAGCTAATTTTGGAGAAGGCTCTGGACCA 221I  W  F  D  D  L  I  C  N  G  N  E  S  A  L  W  N  C  K  H 661ATCTGGTTTGATGATCTTATATGCAACGGAAATGAGTCAGCTCTCTGGAACTGCAAACAT 241Q  G  W  G  K  H  N  C  D  H  A  E  D  A  G  V  I  C  S  K 721CAAGGATGGGGAAAGCATAACTGTGATCATGCTGAGGATGCTGGAGTGATTTGCTCAAAG 261G  A  D  L  S  L  R  L  V  D  G  V  T  E  C  S  G  R  L  E 781GGAGCAGATCTGAGCCTGAGACTGGTAGATGGAGTCACTGAATGTTCAGGAAGATTAGAA 281V  R  F  Q  G  E  W  G  T  I  C  D  D  G  W  D  S  H  D  A 841GTGAGATTCCAAGGAGAATGGGGGACAATATGTGATGACGGCTGGGACAGTCATGATGCT 301A  V  A  C  K  Q  L  G  C  P  T  A  I  T  A  I  G  R  V  N 901GCTGTGGCATGCAAGCAACTGGGATGTCCAACTGCTATCACCGCCATTGGTCGAGTTAAC 321A  S  E  G  F  G  H  I  W  L  D  S  V  S  C  Q  G  H  E  P 961GCCAGTGAGGGATTTGGACACATCTGGCTTGACAGTGTTTCTTGCCAGGGACATGAACCT 341A  V  W  Q  C  K  H  H  E  W  G  K  H  Y  C  N  H  N  E  D 1021GCGGTCTGGCAATGTAAACACCATGAATGGGGAAAGCATTATTGCAATCACAATGAAGAT 361A  G  V  T  C  S  D  G  S  D  L  E  L  R  L  R  G  G  G  S 1081GCTGGCGTAACATGTTCTGATGGATCAGATCTGGAGCTAAGACTTAGAGGTGGAGGCAGC 381R  C  A  G  T  V  E  V  E  I  Q  R  L  L  G  K  V  C  D  R 1141CGCTGTGCTGGGACAGTTGAGGTGGAGATTCAGAGACTGTTAGGGAAGGTGTGtGACAGA 401G  W  G  L  K  E  A  D  V  V  C  R  Q  L  G  C  G  S  A  L 1201GGCTGGGGACTGAAAGAAGCTGATGTGGTTTGCAGGCAGCTGGGATGTGGATCTGCACTC 421K  T  S  Y  Q  V  Y  S  K  I  Q  A  T  N  M  W  L  F  L  S 1261AAAACATCCTATCAAGTATACTCCAAAATCCAGGCAACAAACATGTGGCTGTTTCTAAGT 441S  C  N  G  N  E  T  S  L  W  D  C  K  N  W  Q  W  G  G  L 1321AGCTGTAACGGAAATGAAACTTCTCTTTGGGACTGCAAGAACTGGCAATGGGGTGGACTT 461T  C  D  H  Y  E  E  A  K  I  T  C  S  A  H  R  E  P  R  L 1381ACCTGTGATCACTATGAAGAAGCCAAAATTACCTGCTCAGCCCACAGGGAACCCAGACTG 481V  G  G  D  I  P  C  S  G  R  V  E  V  K  H  G  D  T  W  G 1441GTTGGAGGAGACATTCCCTGTTCTGGACGCGTTGAAGTGAAGCATGGTGACACATGGGGC 501S  V  C  D  S  D  F  S  L  E  A  A  S  V  L  C  R  E  L  Q 1501TCCGTCTGTGATTCGGATTTCTCTCTGGAAGCTGCCAGCGTTCTATGCAGGGAATTACAG 521C  G  T  V  V  S  I  L  G  G  A  H  F  G  E  G  N  G  Q  I 1561TGTGGCACAGTCGTCTCTATCCTGGGGGGAGCTCACTTTGGAGAGGGAAATGGACAGATC 541W  A  E  E  F  Q  C  E  G  H  E  S  H  L  S  L  C  P  V  A 1621TGGGCTGAAGAATTCCAGTGTGAGGGACATGAGTCCCATCTTTCACTCTGCCCAGTAGCA 561P  R  P  E  G  T  C  S  H  S  R  D  V  G  V  V  C  S  R  Y 1681CCCCGCCCAGAAGGAACTTGTAGCCACAGCAGGGATGTTGGAGTAGTCTGCTCAAGATAC 581T  E  I  R  L  V  N  G  K  T  P  C  E  G  R  V  E  L  K  T 1741ACAGAAATTCGCTTGGTGAATGGCAAGACCCCATGTGAGGGCAGAGTGGAGCTCAAAACG 601L  N  A  W  G  S  L  C  N  S  H  W  D  I  E  D  A  H  V  L 1801CTTAATGCCTGGGGATCCCTCTGCAACTCTCACTGGGACATAGAAGATGCCCACGTTCTT 621C  Q  Q  L  K  C  G  V  A  L  S  T  P  G  G  A  H  F  G  K 1861TGCCAACAACTTAAATGTGGAGTTGCCCTTTCTACCCCAGGAGGAGCACATTTTGGAAAA 641G  N  G  Q  V  W  R  H  M  F  H  C  T  G  T  E  Q  H  M  G 1921GGAAATGGTCAGGTCTGGAGGCATATGTTTCACTGCACTGGGACTGAGCAGCACATGGGA 661D  C  P  V  T  A  L  G  A  S  L  C  P  S  G  Q  V  A  S  V 1981GATTGTCCTGTAACTGCTCTGGGTGCTTCACTATGTCCTTCAGGGCAAGTGGCCTCTGTA 681I  C  S  G  N  Q  S  Q  T  L  S  S  C  N  S  S  S  L  G  P 2041ATTTGCTCAGGAAACCAGTCCCAAACACTGTCCTCGTGCAATTCATCATCTCTGGGCCCA 701T  R  P  T  I  P  E  E  S  A  V  A  C  I  E  S  G  Q  L  R 2101ACAAGGCCTACCATTCCAGAAGAAAGTGCTGTGGCCTGCATAGAGAGTGGTCAACTTCGC 721L  V  N  G  G  G  R  C  A  G  R  V  E  I  Y  H  E  G  S  W 2161TTGGTAAATGGAGGAGGTCGCTGTGCTGGGAGAGTAGAGATTTATCATGAGGGCTCCTGG 741G  T  I  C  D  D  S  W  D  L  S  D  A  H  V  V  C  R  Q  L 2221GGCACCATCTGTGATGACAGCTGGGACCTGAGCGATGCCCACGTGGTGTGCAGACAGCTG 761G  C  G  E  A  I  N  A  T  G  S  A  H  F  G  E  G  T  G  P 2281GGCTGTGGAGAGGCCATTAATGCCACTGGTTCTGCTCATTTTGGAGAAGGAACAGGGCCC 781I  W  L  D  E  M  K  C  N  G  K  E  S  R  I  W  Q  C  H  S 2341ATCTGGCTGGATGAGATGAAATGCAATGGAAAAGAATCCCGTATTTGGCAGTGCCATTCA 801H  G  W  G  Q  Q  N  C  R  H  K  E  D  A  G  V  I  C  S  E 2401CATGGCTGGGGGCAGCAAAACTGCAGGCACAAGGAGGATGCAGGAGTTATCTGCTCAGAG 821F  M  S  L  R  L  T  S  E  A  S  R  E  A  C  A  G  R  L  E 2461TTCATGTCTCTGAGACTGACCAGTGAAGCCAGCAGAGAGGCCTGTGCAGGGCGTCTAGAA 841V  F  Y  N  G  A  W  G  S  V  G  R  S  N  M  S  E  T  T  V 2521GTTTTTTACAACGGAGCTTGGGGCAGTGTTGGCAGGAGTAACATGTCTGAAACCACTGTG 861G  V  V  C  R  Q  L  G  C  A  D  K  G  K  I  N  S  A  S  L 2581GGTGTGGTGTGCAGGCAGCTGGGCTGTGCAGACAAAGGGAAAATCAACTCTGCATCTTTA 881D  K  A  M  S  I  P  M  W  V  D  N  V  Q  C  P  K  G  P  D 2641GACAAGGCCATGTCCATTCCCATGTGGGTGGACAATGTTCAGTGTCCAAAAGGACCTGAC 901T  L  W  Q  C  P  S  S  P  W  E  K  R  L  A  R  P  S  E  E 2701ACGCTGTGGCAGTGCCCATCATCTCCATGGGAGAAGAGACTGGCCAGGCCCTCGGAGGAG 921T  W  I  T  C  D  N  K  M  R  L  Q  E  G  P  T  S  C  S  G 2761ACCTGGATCACATGTGACAACAAGATGAGACTACAAGAAGGACCCACTTCCTGTTCTGGA 941R  V  E  I  W  H  G  G  S  W  G  T  V  C  D  D  S  W  D  L 2821CGTGTGGAGATCTGGCACGGAGGTTCCTGGGGGACAGTGTGTGATGACTCCTGGGACTTG 961N  D  A  Q  V  V  C  Q  Q  L  G  C  G  P  A  L  K  A  F  K 2881AACGATGCTCAGGTGGTGTGTCAACAACTTGGCTGTGGTCCAGCTTTGAAAGCATTCAAA 981E  A  E  F  G  Q  G  T  G  P  I  W  L  N  E  V  K  C  K  G 2941GAAGCAGAGTTTGGTCAGGGGACTGGACCCATATGGCTCAATGAAGTGAAGTGCAAAGGG 1001N  E  S  S  L  W  D  C  P  A  R  R  W  G  H  S  E  C  G  H 3001AATGAGTCTTCCTTGTGGGATTGTCCTGCCAGACGCTGGGGCCACAGTGAGTGTGGACAC 1021K  E  D  A  A  V  N  C  T  A  Q  K  I  S  T  H  K  T  P  Q 3061AAGGAAGACGCTGCAGTGAATTGCACAGCACAAAAAATTTCAACGCACAAAACCCCACAA 1041K  A  T  T  G  R  S  F  L  I  A  F  G  I  L  G  V  V  L  L 3121AAAGCCACAACAGGTCGGTCATTCCTTATTGCATTCGGAATCCTTGGAGTTGTTCTCTTG 1061A  I  F  V  A  L  F  L  T  Q  K  R  R  Q  R  Q  R  L  T  V 3181GCCATTTTCGTCGCATTATTCTTGACTCAAAAGCGAAGACAGAGACAGCGGCTTACAGTT 1081S  S  R  G  E  N  L  V  H  Q  I  Q  Y  R  E  M  N  S  C  L 3241TCCTCAAGAGGAGAGAACTTAGTCCACCAAATTCAATACCGGGAGATGAATTCTTGCCTG 1101N  A  D  D  L  D  L  M  N  S  S  G  G  H  S  E  A  H 3301AATGCAGATGATCTGGACCTAATGAATTCCTCAGGAGGCCATTCTGAGGCACAC 1MSKLRMVLLE DSGSADVRRH FVNLSPFTIA VVLLLRACFV TSSLGGTTKE LRLVDGENKC SEQ ID61 SGRVEVKIQE EWGTVCNNGW SMEAVSVICN QLGCPTAIKA TGWANSSAGS GRIWMDHVSCNO: 42 121RGNESALWDC KHDGWGKHSN CTHQQDAGVT CSDGSDLEMR LTNGGNMCSG RIEIKFQGQW 181GTVCDDNFNI NHASVVCKQL ECGSAVSFSG SANFGEGSGP IWFDDLICNG NESALWNCKH 241QGWGKHNCDH AEDAGVICSK GADLSLRLVD GVTECSGRLE VRFQGEWGTI CDDGWDSHDA 301AVACKQLGCP TAITAIGRVN ASEGFGHIWL DSVSCQGHEP AVWQCKHHEW GKHYCNHNED 361AGVTCSDGSD LELRLRGGGS RCAGTVEVEI QRLLGKVCDR GWGLKEADVV CRQLGCGSAL 421KTSYQVYSKI QATNMWLFLS SCNGNETSLW DCKNWQWGGL TCDHYEEAKI TCSAHREPRL 481VGGDIPCSGR VEVKHGDTWG SVCDSDFSLE AASVLCRELQ CGTVVSILGG AHFGEGNGQI 541WAEEFQCEGH ESHLSLCPVA PRPEGTCSHS RDVGVVCSRY TEIRLVNGKT PCEGRVELKT 601LNAWGSLCNS HWDIEDAHVL CQQLKCGVAL STPGGAHFGK GNGQVWRHMF HCTGTEQHMG 661DCPVTALGAS LCPSGQVASV ICSGNQSQTL SSCNSSSLGP TRPTIPEESA VACIESGQLR 721LVNGGGRCAG RVEIYHEGSW GTICDDSWDL SDAHVVCRQL GCGEAINATG SAHFGEGTGP 781IWLDEMKCNG KESRIWQCHS HGWGQQNCRH KEDAGVICSE FMSLRLTSEA SREACAGRLE 841VFYNGAWGSV GRSNMSETTV GVVCRQLGCA DKGKINSASL DKAMSIPMWV DNVQCPKGPD 901TLWQCPSSPW EKRLARPSEE TWITCDNKMR LQEGPTSCSG RVEIWHGGSW GTVCDDSWDL 961NDAQVVCQQL GCGPALKAFK EAEFGQGTGP IWLNEVKCKG NESSLWDCPA RRWGHSECGH 1021KEDAAVNCTA QKISTHKTPQ KATTGRSFLI AFGILGVVLL AIFVALFLTQ KRRQRQRLTV 1081SSRGENLVHQ IQYREMNSCL NADDLDLMNS SGGHSEAH 1M  S  K  L  R  M  V  L  L  E  D  S  G  S  A  D  V  R  R  H SEQ ID 1ATGAGCAAACTCAGAATGGTGCTACTTGAAGACTCTGGATCTGCTGACGTCAGAAGACAT NO: 43 21F  V  N  L  S  P  F  T  I  A  V  V  L  L  L  R  A  C  F  V and 44 61TTTGTCAACTTGAGTCCCTTCACTATTGCTGTGGTCTTACTTCTCCGTGCCTGTTTTGTC 41T  S  S  L  G  G  T  T  K  E  L  R  L  V  D  G  E  N  K  C 121ACCAGTTCTCTTGGAGGAACAACCAAGGAGCTGAGGCTAGTGGATGGTGAAAACAAGTGT 61S  G  R  V  E  V  K  I  Q  E  E  W  G  T  V  C  N  N  G  W 181AGTGGGAGAGTGGAAGTGAAAATCCAGGAGGAGTGGGGAACGGTGTGTAATAATGGCTGG 81S  M  E  A  V  S  V  I  C  N  Q  L  G  C  P  T  A  I  K  A 241AGCATGGAAGCAGTCTCTGTGATTTGTAACCAGCTGGGATGTCCAACTGCTATCAAAGCC 101T  G  W  A  N  S  S  A  G  S  G  R  I  W  M  D  H  V  S  C 301ACTGGATGGGCTAATTCCAGTGCAGGTTCTGGACGCATTTGGATGGATCATGTTTCTTGT 121R  G  N  E  S  A  L  W  D  C  K  H  D  G  W  G  K  H  S  N 361CGTGGGAATGAGTCAGCTCTTTGGGACTGCAAACATGATGGATGGGGAAAGCATAGTAAC 141C  T  H  Q  Q  D  A  G  V  T  C  S  D  G  S  D  L  E  M  R 421TGTACTCACCAACAAGATGCTGGAGTGACTTGCTCAGATGGATCCGATTTGGAAATGAGG 161L  T  N  G  G  N  M  C  S  G  R  I  E  I  K  F  Q  G  Q  W 481CTGACGAATGGAGGGAATATGTGTTCTGGAAGAATAGAGATCAAATTCCAAGGACAGTGG 181G  T  V  C  D  D  N  F  N  I  N  H  A  S  V  V  C  K  Q  L 541GGAACAGTGTGTGATGATAACTTCAACATCAATCATGCATCTGTGGTTTGTAAACAACTT 201E  C  G  S  A  V  S  F  S  G  S  A  N  F  G  E  G  S  G  P 601GAATGTGGAAGTGCTGTCAGTTTCTCTGGTTCAGCTAATTTTGGAGAAGGCTCTGGACCA 221I  W  F  D  D  L  I  C  N  G  N  E  S  A  L  W  N  C  K  H 661ATCTGGTTTGATGATCTTATATGCAACGGAAATGAGTCAGCTCTCTGGAACTGCAAACAT 241Q  G  W  G  K  H  N  C  D  H  A  E  D  A  G  V  I  C  S  K 721CAAGGATGGGGAAAGCATAACTGTGATCATGCTGAGGATGCTGGAGTGATTTGCTCAAAG 261G  A  D  L  S  L  R  L  V  D  G  V  T  E  C  S  G  R  L  E 781GGAGCAGATCTGAGCCTGAGACTGGTAGATGGAGTCACTGAATGTTCAGGAAGATTAGAA 281V  R  F  Q  G  E  W  G  T  I  C  D  D  G  W  D  S  H  D  A 841GTGAGATTCCAAGGAGAATGGGGGACAATATGTGATGACGGCTGGGACAGTCATGATGCT 301A  V  A  C  K  Q  L  G  C  P  T  A  I  T  A  I  G  R  V  N 901GCTGTGGCATGCAAGCAACTGGGATGTCCAACTGCTATCACCGCCATTGGTCGAGTTAAC 321A  S  E  G  F  G  H  I  W  L  D  S  V  S  C  Q  G  H  E  P 961GCCAGTGAGGGATTTGGACACATCTGGCTTGACAGTGTTTCTTGCCAGGGACATGAACCT 341A  V  W  Q  C  K  H  H  E  W  G  K  H  Y  C  N  H  N  E  D 1021GCGGTCTGGCAATGTAAACACCATGAATGGGGAAAGCATTATTGCAATCACAATGAAGAT 361A  G  V  T  C  S  D  G  S  D  L  E  L  R  L  R  G  G  G  S 1081GCTGGCGTAACATGTTCTGATGGATCAGATCTGGAGCTAAGACTTAGAGGTGGAGGCAGC 381R  C  A  G  T  V  E  V  E  I  Q  R  L  L  G  K  V  C  D  R 1141CGCTGTGCTGGGACAGTTGAGGTGGAGATTCAGAGACTGTTAGGGAAGGTGTGTGACAGA 401G  W  G  L  K  E  A  D  V  V  C  R  Q  L  G  C  G  S  A  L 1201GGCTGGGGACTGAAAGAAGCTGATGTGGTTTGCAGGCAGCTGGGATGTGGATCTGCACTC 421K  T  S  Y  Q  V  Y  S  K  I  Q  A  T  N  M  W  L  F  L  S 1261AAAACATCCTATCAAGTATACTCCAAAATCCAGGCAACAAACATGTGGCTGTTTCTAAGT 441S  C  N  G  N  E  T  S  L  W  D  C  K  N  W  Q  W  G  G  L 1321AGCTGTAACGGAAATGAAACTTCTCTTTGGGACTGCAAGAACTGGCAATGGGGTGGACTT 461T  C  D  H  Y  E  E  A  K  I  T  C  S  A  H  R  E  P  R  L 1381ACCTGTGATCACTATGAAGAAGCCAAAATTACCTGCTCAGCCCACAGGGAACCCAGACTG 481V  G  G  D  I  P  C  S  G  R  V  E  V  K  H  G  D  T  W  G 1441GTTGGAGGAGACATTCCCTGTTCTGGACGCGTTGAAGTGAAGCATGGTGACACATGGGGC 501S  V  C  D  S  D  F  S  L  E  A  A  S  V  L  C  R  E  L  Q 1501TCCGTCTGTGATTCGGATTTCTCTCTGGAAGCTGCCAGCGTTCTATGCAGGGAATTACAG 521C  G  T  V  V  S  I  L  G  G  A  H  F  G  E  G  N  G  Q  I 1561TGTGGCACAGTCGTCTCTATCCTGGGGGGAGCTCACTTTGGAGAGGGAAATGGACAGATC 541W  A  E  E  F  Q  C  E  G  H  E  S  H  L  S  L  C  P  V  A 1621TGGGCTGAAGAATTCCAGTGTGAGGGACATGAGTCCCATCTTTCACTCTGCCCAGTAGCA 561P  R  P  E  G  T  C  S  H  S  R  D  V  G  V  V  C  S  R  Y 1681CCCCGCCCAGAAGGAACTTGTAGCCACAGCAGGGATGTTGGAGTAGTCTGCTCAAGATAC 581T  E  I  R  L  V  N  G  K  T  P  C  E  G  R  V  E  L  K  T 1741ACAGAAATTCGCTTGGTGAATGGCAAGACCCCATGTGAGGGCAGAGTGGAGCTCAAAACG 601L  N  A  W  G  S  L  C  N  S  H  W  D  I  E  D  A  H  V  L 1801CTTAATGCCTGGGGATCCCTCTGCAACTCTCACTGGGACATAGAAGATGCCCACGTTCTT 621C  Q  Q  L  K  C  G  V  A  L  S  T  P  G  G  A  H  F  G  K 1861TGCCAACAACTTAAATGTGGAGTTGCCCTTTCTACCCCAGGAGGAGCACATTTTGGAAAA 641G  N  G  Q  V  W  R  H  M  F  H  C  T  G  T  E  Q  H  M  G 1921GGAAATGGTCAGGTCTGGAGGCATATGTTTCACTGCACTGGGACTGAGCAGCACATGGGA 661D  C  P  V  T  A  L  G  A  S  L  C  P  S  G  Q  V  A  S  V 1981GATTGTCCTGTAACTGCTCTGGGTGCTTCACTATGTCCTTCAGGGCAAGTGGCCTCTGTA 681I  C  S  G  N  Q  S  Q  T  L  S  S  C  N  S  S  S  L  G  P 2041ATTTGCTCAGGAAACCAGTCCCAAACACTGTCCTCGTGCAATTCATCATCTCTGGGCCCA 701T  R  P  T  I  P  E  E  S  A  V  A  C  I  E  S  G  Q  L  R 2101ACAAGGCCTACCATTCCAGAAGAAAGTGCTGTGGCCTGCATAGAGAGTGGTCAACTTCGC 721L  V  N  G  G  G  R  C  A  G  R  V  E  I  Y  H  E  G  S  W 2161TTGGTAAATGGAGGAGGTCGCTGTGCTGGGAGAGTAGAGATTTATCATGAGGGCTCCTGG 741G  T  I  C  D  D  S  W  D  L  S  D  A  H  V  V  C  R  Q  L 2221GGCACCATCTGTGATGACAGCTGGGACCTGAGCGATGCCCACGTGGTGTGCAGACAGCTG 761G  C  G  E  A  I  N  A  T  G  S  A  H  F  G  E  G  T  G  P 2281GGCTGTGGAGAGGCCATTAATGCCACTGGTTCTGCTCATTTTGGAGAAGGAACAGGGCCC 781I  W  L  D  E  M  K  C  N  G  K  E  S  R  I  W  Q  C  H  S 2341ATCTGGCTGGATGAGATGAAATGCAATGGAAAAGAATCCCGTATTTGGCAGTGCCATTCA 801H  G  W  G  Q  Q  N  C  R  H  K  E  D  A  G  V  I  C  S  E 2401CATGGCTGGGGGCAGCAAAACTGCAGGCACAAGGAGGATGCAGGAGTTATCTGCTCAGAG 821F  M  S  L  R  L  T  S  E  A  S  R  E  A  C  A  G  R  L  E 2461TTCATGTCTCTGAGACTGACCAGTGAAGCCAGCAGAGAGGCCTGTGCAGGGCGTCTAGAA 841V  F  Y  N  G  A  W  G  S  V  G  R  S  N  M  S  E  T  T  V 2521GTTTTTTACAACGGAGCTTGGGGCAGTGTTGGCAGGAGTAACATGTCTGAAACCACTGTG 861G  V  V  C  R  Q  L  G  C  A  D  K  G  K  I  N  S  A  S  L 2581GGTGTGGTGTGCAGGCAGCTGGGCTGTGCAGACAAAGGGAAAATCAACTCTGCATCTTTA 881D  K  A  M  S  I  P  M  W  V  D  N  V  Q  C  P  K  G  P  D 2641GACAAGGCCATGTCCATTCCCATGTGGGTGGACAATGTTCAGTGTCCAAAAGGACCTGAC 901T  L  W  Q  C  P  S  S  P  W  E  K  R  L  A  R  P  S  E  E 2701ACGCTGTGGCAGTGCCCATCATCTCCATGGGAGAAGAGACTGGCCAGGCCCTCGGAGGAG 921T  W  I  T  C  D  N  K  M  R  L  Q  E  G  P  T  S  C  S  G 2761ACCTGGATCACATGTGACAACAAGATGAGACTACAAGAAGGACCCACTTCCTGTTCTGGA 941R  V  E  I  W  H  G  G  S  W  G  T  V  C  D  D  S  W  D  L 2821CGTGTGGAGATCTGGCACGGAGGTTCCTGGGGGACAGTGTGTGATGACTCCTGGGACTTG 961N  D  A  Q  V  V  C  Q  Q  L  G  C  G  P  A  L  K  A  F  K 2881AACGATGCTCAGGTGGTGTGTCAACAACTTGGCTGTGGTCCAGCTTTGAAAGCATTCAAA 981E  A  E  F  G  Q  G  T  G  P  I  W  L  N  E  V  K  C  K  G 2941GAAGCAGAGTTTGGTCAGGGGACTGGACCCATATGGCTCAATGAAGTGAAGTGCAAAGGG 1001N  E  S  S  L  W  D  C  P  A  R  R  W  G  H  S  E  C  G  H 3001AATGAGTCTTCCTTGTGGGATTGTCCTGCCAGACGCTGGGGCCACAGTGAGTGTGGACAC 1021K  E  D  A  A  V  N  C  T  A  Q  K  I  S  T  H  K  T  P  Q 3061AAGGAAGACGCTGCAGTGAATTGCACAGCACAAAAAATTTCAACGCACAAAACCCCACAA 1041K  A  T  T  G  Q  S  F  L  I  A  F  G  I  L  G  V  V  L  L 3121AAAGCCACAACAGGTCAGTCATTCCTTATTGCATTCGGAATCCTTGGAGTTGTTCTCTTG 1061A  I  F  V  A  L  F  L  T  Q  K  R  R  Q  R  Q  R  L  T  V 3181GCCATTTTCGTCGCATTATTCTTGACTCAAAAGCGAAGACAGAGACAGCGGCTTACAGTT 1081S  S  R  G  E  N  L  V  H  Q  I  Q  Y  R  E  M  N  S  C  L 3241TCCTCAAGAGGAGAGAACTTAGTCCACCAAATTCAATACCGGGAGATGAATTCTTGCCTG 1101N  A  D  D  L  D  L  M  N  S  S  E  N  S  N  E  S  A  D  F 3301AATGCAGATGATCTGGACCTAATGAATTCCTCAGAAAATTCCAATGAGTCAGCTGATTTC 1121N  A  A  E  L  I  S  V  S  K  F  L  P  I  S  G  M  E  K  E 3361AATGCTGCTGAACTAATTTCTGTGTCTAAATTTCTTCCTATTTCTGGAATGGAAAAGGAG 1141A  I  L  R  H  T  E  K  E  N  G  N  L 3421GCCATTCTGAGGCACACTGAAAAGGAAAATGGGAATTTA 1MSKLRMVLLE DSGSADVRRH FVNLSPFTIA VVLLLRACFV TSSLGGTTKE LRLVDGENKC SEQ ID61 SGRVEVKIQE EWGTVCNNGW SMEAVSVICN QLGCPTAIKA TGWANSSAGS GRIWMDHVSCNO: 44 121RGNESALWDC KHDGWGKHSN CTHQQDAGVT CSDGSDLEMR LTNGGNMCSG RIEIKFQGQW 181GTVCDDNFNI NHASVVCKQL ECGSAVSFSG SANFGEGSGP IWFDDLICNG NESALWNCKH 241QGWGKHNCDH AEDAGVICSK GADLSLRLVD GVTECSGRLE VRFQGEWGTI CDDGWDSHDA 301AVACKQLGCP TAITAIGRVN ASEGFGHIWL DSVSCQGHEP AVWQCKHHEW GKHYCNHNED 361AGVTCSDGSD LELRLRGGGS RCAGTVEVEI QRLLGKVCDR GWGLKEADVV CRQLGCGSAL 421KTSYQVYSKI QATNMWLFLS SCNGNETSLW DCKNWQWGGL TCDHYEEAKI TCSAHREPRL 481VGGDIPCSGR VEVKHGDTWG SVCDSDFSLE AASVLCRELQ CGTVVSILGG AHFGEGNGQI 541WAEEFQCEGH ESHLSLCPVA PRPEGTCSHS RDVGVVCSRY TEIRLVNGKT PCEGRVELKT 601LNAWGSLCNS HWDIEDAHVL CQQLKCGVAL STPGGAHFGK GNGQVWRHMF HCTGTEQHMG 661DCPVTALGAS LCPSGQVASV ICSGNQSQTL SSCNSSSLGP TRPTIPEESA VACIESGQLR 721LVNGGGRCAG RVEIYHEGSW GTICDDSWDL SDAHVVCRQL GCGEAINATG SAHFGEGTGP 781IWLDEMKCNG KESRIWQCHS HGWGQQNCRH KEDAGVICSE FMSLRLTSEA SREACAGRLE 841VFYNGAWGSV GRSNMSETTV GVVCRQLGCA DKGKINSASL DKAMSIPMWV DNVQCPKGPD 901TLWQCPSSPW EKRLARPSEE TWITCDNKMR LQEGPTSCSG RVEIWHGGSW GTVCDDSWDL 961NDAQVVCQQL GCGPALKAFK EAEFGQGTGP IWLNEVKCKG NESSLWDCPA RRWGHSECGH 1021KEDAAVNCTA QKISTHKTPQ KATTGQSFLI AFGILGVVLL AIFVALFLTQ KRRQRQRLTV 1081SSRGENLVHQ IQYREMNSCL NADDLDLMNS SENSNESADF NAAELISVSK FLPISGMEKE 1141AILRHTEKEN GNL

Example 10 Cloning and Characterization of Canine CD163 from DH82 Cells

A forward primer 5′ simianCD163 (SEQ ID NO: 28)(5′-CACCGGAATGAGCAAACTCAGAATGG-3′ based on human CD163) and a reverseprimer HuCD163-3′Kpn (SEQ ID NO: 29) (5′-GCTCCGGTACCTAGTCCAGGTCTTCATCAAGGTATCTTA-3′) were used to amplify CD163 cDNA from DH82 cells. Totalcellular RNA was prepared from DH82 cells using the RNeasy kit. RT-PCRparameters were the same as described in Example 4. RT-PCR products werecloned directionally into the pcDNA3.1D/V5/His/TOPO vector according tothe manufacturer's instruction. Several clones containing large insertswere analyzed. Several clones with large inserts were analyzed, andthese fell into either the v2 or v3 splicing patterns seen in otherspecies. The v2 variant is missing an 81-nucleotide exon (E81) relativeto the v3 variant, which results in a reading frame shift andalternative carboxy terminal amino acid sequences. The canine CD163v2cDNA from DH82 cells encodes a peptide of 1115 amino acids. Whencompared to the sequences in Genbank database, it is 83.9% identical tohuman CD163 (Genbank Z22968), 85.1% identical to pig CD163 (GenbankAJ311716), and 74.3% identical to mouse CD163 (Genbank AF274883). Thenucleotide and amino acid sequences of the two splice variants found inDH82 cells are provided below (SEQ ID NOS: 45-48).

SEQUENCE ID NO 1M  S  K  L  R  M  V  P  H  G  N  S  G  S  A  D  F  R  R  C SEQ ID 1ATGAGCAAACTCAGAATGGTCCCACATGGAAACTCTGGATCTGCTGACTTTAGAAGATGT NO: 45 21F  A  L  L  C  P  S  A  V  A  V  V  S  I  L  S  T  C  L  M and 46 61TTTGCCCTCTTGTGTCCCTCTGCTGTGGCTGTGGTCTCCATTCTCAGTACCTGTTTGATG 41T  N  S  L  G  R  A  D  K  E  M  R  L  T  D  G  E  D  N  C 121ACCAATTCTCTTGGGAGAGCAGATAAAGAGATGAGGCTAACGGATGGTGAAGACAATTGC 61S  G  R  V  E  V  K  V  Q  E  E  W  G  T  V  C  N  N  G  W 181TCCGGGAGAGTGGAAGTGAAAGTCCAGGAGGAGTGGGGAACGGTGTGTAATAATGGCTGG 81G  M  D  E  V  S  V  I  C  R  Q  L  G  C  P  T  A  I  K  A 241GGCATGGATGAAGTCTCTGTGATTTGCAGGCAGCTGGGATGTCCCACTGCTATCAAAGCC 101A  G  W  A  N  S  R  A  G  S  G  R  I  W  M  D  H  V  S  C 301GCTGGATGGGCCAATTCCAGGGCAGGCTCTGGACGAATCTGGATGGATCATGTTTCTTGT 121R  G  N  E  S  A  L  W  D  C  K  H  D  G  W  G  K  H  N  C 361CGAGGGAATGAATCTGCTCTCTGGGACTGCAAACATGATGGATGGGGAAAGCACAACTGC 141S  H  Q  Q  D  A  G  V  T  C  S  D  G  S  S  L  E  M  R  L 421AGTCATCAACAGGATGCTGGAGTAACCTGTTCAGATGGATCCAGTTTGGAGATGAGGTTG 161M  N  G  G  N  Q  C  S  G  R  I  E  V  K  F  Q  G  Q  W  G 481ATGAACGGCGGAAACCAGTGTTCTGGCAGAATAGAAGTCAAGTTCCAGGGACAGTGGGGA 181T  V  C  D  D  N  F  N  I  D  H  A  S  V  V  C  K  Q  L  E 541ACAGTGTGTGATGACAACTTCAACATAGATCATGCTTCTGTGGTTTGTAAACAGCTCGAA 201C  G  S  A  V  S  F  S  G  S  A  N  F  G  E  G  S  G  P  I 601TGTGGAAGTGCTGTCAGTTTCTCTGGTTCAGCTAATTTTGGAGAAGGTTCTGGGCCAATC 221W  F  D  D  L  V  C  S  G  N  E  S  A  L  W  N  C  K  H  E 661TGGTTTGATGATCTTGTGTGCAGTGGAAATGAGTCAGCTCTCTGGAACTGCAAGCATGAA 241G  W  G  K  H  N  C  D  H  A  E  D  V  G  V  I  C  L  D  G 721GGATGGGGAAAGCATAACTGTGATCACGCTGAGGATGTTGGAGTGATTTGCTTGGATGGA 261A  D  L  S  L  R  L  V  D  G  V  T  E  C  S  G  R  L  E  V 781GCAGATCTGAGCCTGAGACTGGTAGATGGAGTCACTGAATGTTCAGGAAGATTAGAAGTA 281K  F  Q  G  E  W  G  T  V  C  D  D  G  W  D  S  N  D  A  A 841AAATTCCAAGGGGAATGGGGGACAGTGTGTGATGATGGCTGGGATAGTAATGATGCTGCT 301V  V  C  K  Q  L  G  C  P  T  A  V  T  A  I  G  R  V  N  A 901GTGGTATGTAAACAACTGGGATGCCCAACTGCTGTCACCGCCATTGGTCGAGTTAACGCC 321S  E  G  S  G  H  I  W  L  D  N  L  S  C  Q  G  D  E  S  A 961AGTGAGGGAAGTGGACACATTTGGCTTGACAATCTTTCCTGCCAAGGAGACGAATCTGCT 341L  W  Q  C  R  H  H  E  W  G  K  H  Y  C  N  H  N  E  D  A 1021CTCTGGCAGTGTAGACACCATGAATGGGGAAAGCATTATTGCAATCATAATGAAGATGCT 361G  V  T  C  S  D  G  S  D  L  E  L  R  L  V  G  G  G  S  R 1081GGTGTGACATGTTCTGATGGATCAGACCTGGAGCTGAGACTTGTCGGTGGAGGCAGCCGC 381C  A  G  T  V  E  V  E  I  Q  K  L  L  G  K  V  C  D  R  G 1141TGTGCTGGGACAGTGGAGGTTGAAATTCAGAAACTGCTAGGGAAAGTATGTGATAGAGGC 401W  G  L  K  E  A  D  V  V  C  K  Q  L  G  C  G  S  A  L  K 1201TGGGGACTGAAAGAAGCCGATGTGGTTTGCAAGCAGTTGGGATGTGGATCTGCTCTCAAA 421T  S  Y  Q  R  Y  S  K  V  K  A  T  N  T  W  L  F  L  S  R 1261ACGTCCTATCAGCGTTATTCCAAAGTTAAGGCAACAAACACATGGCTGTTTTTAAGCCGC 441C  S  G  N  E  T  S  L  W  D  C  K  N  W  Q  W  G  G  L  S 1321TGTAGTGGCAATGAAACTTCCCTTTGGGACTGCAAGAACTGGCAGTGGGGTGGACTGAGC 461C  D  H  Y  E  E  A  K  V  T  C  S  A  H  R  E  P  R  L  V 1381TGTGATCACTATGAAGAAGCTAAAGTTACCTGCTCAGCCCACAGGGAACCCAGACTAGTT 481G  G  D  I  P  C  S  G  R  V  E  V  K  H  G  D  T  W  G  T 1441GGAGGAGATATTCCCTGCTCTGGTCGTGTTGAAGTGAAACATGGTGACACATGGGGCACC 501V  C  D  S  D  F  S  L  E  A  A  S  V  L  C  R  E  L  Q  C 1501GTCTGTGATTCCGACTTCTCTTTGGAAGCTGCCAGTGTGCTGTGCAGAGAGTTACAGTGT 521G  T  V  I  S  I  L  G  G  A  H  F  G  E  G  N  G  Q  I  W 1561GGCACAGTCATCTCCATCCTAGGGGGAGCTCACTTTGGAGAAGGAAATGGACAGATCTGG 541A  E  E  F  Q  C  E  G  Q  E  S  H  L  S  L  C  S  V  A  S 1621GCTGAAGAATTCCAGTGTGAGGGGCAGGAGTCCCATCTTTCACTCTGTTCAGTAGCCTCT 561R  P  D  G  T  C  S  H  S  R  D  V  G  V  V  C  S  R  Y  T 1681CGCCCAGATGGGACCTGTAGCCACAGCAGGGATGTTGGAGTCGTCTGCTCAAGATACACG 581E  I  R  L  V  N  G  Q  S  P  C  E  G  R  V  E  L  K  I  L 1741GAAATCCGCTTGGTGAATGGCCAGTCCCCGTGTGAAGGAAGAGTGGAGCTCAAGATACTT 601G  N  W  G  S  L  C  N  S  H  W  D  I  E  D  A  H  V  F  C 1801GGGAACTGGGGATCCCTCTGCAACTCTCACTGGGACATAGAAGATGCCCATGTTTTCTGT 621Q  Q  L  K  C  G  V  A  L  S  I  P  G  G  A  H  F  G  K  G 1861CAGCAGCTCAAATGTGGAGTTGCCCTTTCTATTCCGGGAGGAGCACATTTTGGGAAAGGA 641S  G  Q  I  W  R  H  M  F  H  C  T  G  T  E  Q  H  M  G  D 1921AGTGGTCAGATCTGGAGGCACATGTTTCACTGCACTGGGACTGAGCAGCACATGGGAGAT 661C  P  V  T  A  L  G  A  T  L  C  S  A  G  Q  V  A  S  V  I 1981TGCCCTGTAACTGCTCTGGGCGCGACGCTGTGTTCTGCTGGGCAAGTGGCCTCTGTAATC 681C  S  G  N  Q  S  Q  T  L  S  P  C  N  S  T  S  L  D  P  T 2041TGCTCAGGAAATCAGAGCCAGACGCTATCCCCATGCAATTCAACATCTCTGGACCCAACA 701R  S  T  T  S  E  E  S  A  V  A  C  I  A  S  G  Q  L  R  L 2101AGATCTACCACTTCGGAAGAAAGTGCTGTTGCTTGTATTGCGAGTGGGCAACTTCGCCTG 721V  N  G  G  G  R  C  A  G  R  I  E  V  Y  H  E  G  S  W  G 2161GTAAATGGAGGCGGTCGCTGTGCTGGGAGAATAGAGGTCTACCATGAGGGCTCCTGGGGC 741T  I  C  D  D  S  W  D  L  S  D  A  H  V  V  C  R  Q  L  G 2221ACCATCTGTGATGACAGCTGGGACCTGAGTGATGCCCATGTGGTGTGCAGACAGCTGGGC 761C  G  V  A  I  N  A  T  G  S  A  H  F  G  E  G  T  G  P  I 2281TGTGGAGTGGCCATTAATGCCACTGGCTCTGCTCATTTTGGGGAAGGAACAGGGCCCATC 781W  L  D  E  V  N  C  N  G  K  E  S  H  I  W  Q  C  R  S  H 2341TGGCTGGACGAGGTGAACTGTAATGGAAAGGAATCTCATATCTGGCAATGCCGCTCACAC 801G  W  G  Q  H  N  C  R  H  K  E  D  A  G  V  I  C  S  E  F 2401GGCTGGGGGCAACACAACTGCAGACATAAGGAGGATGCAGGAGTTATCTGCTCAGAGTTC 821M  S  L  R  L  I  D  E  T  S  R  D  I  C  A  G  R  L  E  V 2461ATGTCTCTCAGACTGATTGATGAAACCAGCAGAGACATCTGTGCAGGGCGTCTTGAAGTT 841F  Y  N  G  A  W  G  S  V  G  K  S  N  M  S  A  T  T  V  E 2521TTTTACAATGGAGCTTGGGGCAGCGTTGGCAAGAGTAATATGTCTGCAACCACTGTGGAG 861V  V  C  R  Q  L  G  C  A  D  K  G  S  I  N  P  A  S  S  D 2581GTGGTATGCAGGCAACTGGGTTGTGCAGACAAGGGGAGCATCAACCCTGCATCTTCAGAC 881K  P  M  S  R  H  M  W  V  D  N  V  Q  C  P  K  G  P  D  T 2641AAGCCCATGTCCAGGCACATGTGGGTGGACAATGTCCAGTGTCCAAAAGGACCTGACACC 901L  W  Q  C  P  S  S  P  W  K  Q  R  V  A  S  S  S  E  E  T 2701TTATGGCAGTGCCCATCTTCTCCATGGAAACAGAGAGTGGCCAGTTCTTCAGAGGAGACC 921W  I  T  C  A  N  K  I  R  L  Q  E  G  T  S  N  C  S  G  R 2761TGGATCACATGTGCCAACAAGATAAGACTTCAAGAAGGAACCTCTAATTGTTCTGGACGT 941V  E  L  W  H  G  G  S  W  G  T  V  C  D  D  S  W  D  L  E 2821GTGGAGCTCTGGCACGGAGGTTCCTGGGGGACAGTGTGCGATGACTCCTGGGACCTTGAA 961D  A  Q  V  V  C  R  Q  L  G  C  G  P  A  L  E  A  L  K  E 2881GATGCACAAGTGGTGTGTCGACAGCTGGGCTGTGGCCCAGCATTAGAAGCACTAAAAGAG 981A  A  F  G  Q  G  T  G  P  I  W  L  N  D  V  K  C  K  G  N 2941GCAGCATTTGGTCAGGGGACTGGGCCTATATGGCTCAATGACGTGAAGTGCAAAGGGAAT 1001E  S  S  L  W  D  C  P  A  R  P  W  G  H  S  D  C  G  H  K 3001GAGTCTTCCTTGTGGGATTGTCCTGCTAGACCCTGGGGGCACAGTGACTGTGGCCACAAG 1021E  D  A  A  V  R  C  S  E  I  A  M  A  Q  R  S  S  N  P  R 3061GAAGATGCTGCTGTGAGGTGCTCAGAAATTGCAATGGCCCAAAGATCATCAAATCCTAGA 1041G  H  S  S  L  V  A  L  G  I  F  G  V  I  L  L  A  F  L  I 3121GGTCACTCATCCCTTGTTGCATTGGGGATCTTTGGTGTCATTCTTCTGGCCTTTCTCATC 1061A  L  L  L  W  T  Q  R  R  R  Q  Q  Q  R  L  T  V  S  L  R 3181GCTCTCCTCTTGTGGACTCAAAGGCGAAGACAGCAACAGCGGCTTACAGTTTCCTTGAGA 1081G  E  N  S  V  H  Q  I  Q  Y  R  E  M  N  S  S  L  K  A  D 3241GGAGAGAATTCTGTCCACCAAATTCAATACCGGGAAATGAATTCTTCCCTGAAAGCAGAT 1101D  L  D  V  L  T  S  S  E  D  H  F  E  V  H 3301GATCTGGACGTGCTGACTTCCTCAGAAGACCATTTTGAGGTACAC 1MSKLRMVPHG NSGSADFRRC FALLCPSAVA VVSILSTCLM TNSLGRADKE MRLTDGEDNC SEQ ID61 SGRVEVKVQE EWGTVCNNGW GMDEVSVICR QLGCPTAIKA AGWANSRAGS GRIWMDHVSCNO: 46 121RGNESALWDC KHDGWGKHNC SHQQDAGVTC SDGSSLEMRL MNGGNQCSGR IEVKFQGQWG 181TVCDDNFNID HASVVCKQLE CGSAVSFSGS ANFGEGSGPI WFDDLVCSGN ESALWNCKHE 241GWGKHNCDHA EDVGVICLDG ADLSLRLVDG VTECSGRLEV KFQGEWGTVC DDGWDSNDAA 301VVCKQLGCPT AVTAIGRVNA SEGSGHIWLD NLSCQGDESA LWQCRHHEWG KHYCNHNEDA 361GVTCSDGSDL ELRLVGGGSR CAGTVEVEIQ KLLGKVCDRG WGLKEADVVC KQLGCGSALK 421TSYQRYSKVK ATNTWLFLSR CSGNETSLWD CKNWQWGGLS CDHYEEAKVT CSAHREPRLV 481GGDIPCSGRV EVKHGDTWGT VCDSDFSLEA ASVLCRELQC GTVISILGGA HFGEGNGQIW 541AEEFQCEGQE SHLSLCSVAS RPDGTCSHSR DVGVVCSRYT EIRLVNGQSP CEGRVELKIL 601GNWGSLCNSH WDIEDAHVFC QQLKCGVALS IPGGAHFGKG SGQIWRHMFH CTGTEQHMGD 661CPVTALGATL CSAGQVASVI CSGNQSQTLS PCNSTSLDPT RSTTSEESAV ACIASGQLRL 721VNGGGRCAGR IEVYHEGSWG TICDDSWDLS DAHVVCRQLG CGVAINATGS AHFGEGTGPI 781WLDEVNCNGK ESHIWQCRSH GWGQHNCRHK EDAGVICSEF MSLRLIDETS RDICAGRLEV 841FYNGAWGSVG KSNMSATTVE VVCRQLGCAD KGSINPASSD KPMSRHMWVD NVQCPKGPDT 901LWQCPSSPWK QRVASSSEET WITCANKIRL QEGTSNCSGR VELWHGGSWG TVCDDSWDLE 961DAQVVCRQLG CGPALEALKE AAFGQGTGPI WLNDVKCKGN ESSLWDCPAR PWGHSDCGHK 1021EDAAVRCSEI AMAQRSSNPR GHSSLVALGI FGVILLAFLI ALLLWTQRRR QQQRLTVSLR 1081GENSVHQIQY REMNSSLKAD DLDVLTSSED HFEVH 1M  S  K  L  R  M  V  P  H  G  N  S  G  S  A  D  F  R  R  C SEQ ID 1ATGAGCAAACTCAGAATGGTCCCACATGGAAACTCTGGATCTGCTGACTTTAGAAGATGT NO: 47 21F  A  L  L  C  P  S  A  V  A  V  V  S  I  L  S  T  C  L  M 61TTTGCCCTCTTGTGTCCCTCTGCTGTGGCTGTGGTCTCCATTCTCAGTACCTGTTTGATG 41T  N  S  L  G  R  A  D  K  E  M  R  L  T  D  G  E  D  N  C 121ACCAATTCTCTTGGGAGAGCAGATAAAGAGATGAGGCTAACGGATGGTGAAGACAATTGC 61S  G  R  V  E  V  K  V  Q  E  E  W  G  T  V  C  N  N  G  W 181TCCGGGAGAGTGGAAGTGAAAGTCCAGGAGGAGTGGGGAACGGTGTGTAATAATGGCTGG 81G  M  D  E  V  S  V  I  C  R  Q  L  G  C  P  T  A  I  K  A 241GGCATGGATGAAGTCTCTGTGATTTGCAGGCAGCTGGGATGTCCCACTGCTATCAAAGCC 101A  G  W  A  N  S  R  A  G  S  G  R  I  W  M  D  H  V  S  C 301GCTGGATGGGCCAATTCCAGGGCAGGCTCTGGACGAATCTGGATGGATCATGTTTCTTGT 121R  G  N  E  S  A  L  W  D  C  K  H  D  G  W  G  K  H  N  C 361CGAGGGAATGAATCTGCTCTCTGGGACTGCAAACATGATGGATGGGGAAAGCACAACTGC 141S  H  Q  Q  D  A  G  V  T  C  S  D  G  S  S  L  E  M  R  L 421AGTCATCAACAGGATGCTGGAGTAACCTGTTCAGATGGATCCAGTTTGGAGATGAGGTTG 161M  N  G  G  N  Q  C  S  G  R  I  E  V  K  F  Q  G  Q  W  G 481ATGAACGGCGGAAACCAGTGTTCTGGCAGAATAGAAGTCAAGTTCCAGGGACAGTGGGGA 181T  V  C  D  D  N  F  N  I  D  H  A  S  V  V  C  K  Q  L  E 541ACAGTGTGTGATGACAACTTCAACATAGATCATGCTTCTGTGGTTTGTAAACAGCTCGAA 201C  G  S  A  V  S  F  S  G  S  A  N  F  G  E  G  S  G  P  I 601TGTGGAAGTGCTGTCAGTTTCTCTGGTTCAGCTAATTTTGGAGAAGGTTCTGGGCCAATC 221W  F  D  D  L  V  C  S  G  N  E  S  A  L  W  N  C  K  H  E 661TGGTTTGATGATCTTGTGTGCAGTGGAAATGAGTCAGCTCTCTGGAACTGCAAGCATGAA 241G  W  G  K  H  N  C  D  H  A  E  D  V  G  V  I  C  L  D  G 721GGATGGGGAAAGCATAACTGTGATCACGCTGAGGATGTTGGAGTGATTTGCTTGGATGGA 261A  D  L  S  L  R  L  V  D  G  V  T  E  C  S  G  R  L  E  V 781GCAGATCTGAGCCTGAGACTGGTAGATGGAGTCACTGAATGTTCAGGAAGATTAGAAGTA 281K  F  Q  G  E  W  G  T  V  C  D  D  G  W  D  S  N  D  A  A 841AAATTCCAAGGGGAATGGGGGACAGTGTGTGATGATGGCTGGGATAGTAATGATGCTGCT 301V  V  C  K  Q  L  G  C  P  T  A  V  T  A  I  G  R  V  N  A 901GTGGTATGTAAACAACTGGGATGCCCAACTGCTGTCACCGCCATTGGTCGAGTTAACGCC 321S  E  G  S  G  H  I  W  L  D  N  L  S  C  Q  G  D  E  S  A 961AGTGAGGGAAGTGGACACATTTGGCTTGACAATCTTTCCTGCCAAGGAGACGAATCTGCT 341L  W  Q  C  R  H  H  E  W  G  K  H  Y  C  N  H  N  E  D  A 1021CTCTGGCAGTGTAGACACCATGAATGGGGAAAGCATTATTGCAATCATAATGAAGATGCT 361G  V  T  C  S  D  G  S  D  L  E  L  R  L  V  G  G  G  S  R 1081GGTGTGACATGTTCTGATGGATCAGACCTGGAGCTGAGACTTGTCGGTGGAGGCAGCCGC 381C  A  G  T  V  E  V  E  I  Q  K  L  L  G  K  V  C  D  R  G 1141TGTGCTGGGACAGTGGAGGTTGAAATTCAGAAACTGCTAGGGAAAGTATGTGATAGAGGC 401W  G  L  K  E  A  D  V  V  C  K  Q  L  G  C  G  S  A  L  K 1201TGGGGACTGAAAGAAGCCGATGTGGTTTGCAAGCAGTTGGGATGTGGATCTGCTCTCAAA 421T  S  Y  Q  R  Y  S  K  V  K  A  T  N  T  W  L  F  L  S  R 1261ACGTCCTATCAGCGTTATTCCAAAGTTAAGGCAACAAACACATGGCTGTTTTTAAGCCGC 441C  S  G  N  E  T  S  L  W  D  C  K  N  W  Q  W  G  G  L  S 1321TGTAGTGGCAATGAAACTTCCCTTTGGGACTGCAAGAACTGGCAGTGGGGTGGACTGAGC 461C  D  H  Y  E  E  A  K  V  T  C  S  A  H  R  E  P  R  L  V 1381TGTGATCACTATGAAGAAGCTAAAGTTACCTGCTCAGCCCACAGGGAACCCAGACTAGTT 481G  G  D  I  P  C  S  G  R  V  E  V  K  H  G  D  T  W  G  T 1441GGAGGAGATATTCCCTGCTCTGGTCGTGTTGAAGTGAAACATGGTGACACATGGGGCACC 501V  C  D  S  D  F  S  L  E  A  A  S  V  L  C  R  E  L  Q  C 1501GTCTGTGATTCCGACTTCTCTTTGGAAGCTGCCAGTGTGCTGTGCAGAGAGTTACAGTGT 521G  T  V  I  S  I  L  G  G  A  H  F  G  E  G  N  G  Q  I  W 1561GGCACAGTCATCTCCATCCTAGGGGGAGCTCACTTTGGAGAAGGAAATGGACAGATCTGG 541A  E  E  F  Q  C  E  G  Q  E  S  H  L  S  L  C  S  V  A  S 1621GCTGAAGAATTCCAGTGTGAGGGGCAGGAGTCCCATCTTTCACTCTGTTCAGTAGCCTCT 561R  P  D  G  T  C  S  H  S  R  D  V  G  V  V  C  S  R  Y  T 1681CGCCCAGATGGGACCTGTAGCCACAGCAGGGATGTTGGAGTCGTCTGCTCAAGATACACG 581E  I  R  L  V  N  G  Q  S  P  C  E  G  R  V  E  L  K  I  L 1741GAAATCCGCTTGGTGAATGGCCAGTCCCCGTGTGAAGGAAGAGTGGAGCTCAAGATACTT 601G  N  W  G  S  L  C  N  S  H  W  D  I  E  D  A  H  V  F  C 1801GGGAACTGGGGATCCCTCTGCAACTCTCACTGGGACATAGAAGATGCCCATGTTTTCTGT 621Q  Q  L  K  C  G  V  A  L  S  I  P  G  G  A  H  F  G  K  G 1861CAGCAGCTCAAATGTGGAGTTGCCCTTTCTATTCCGGGAGGAGCACATTTTGGGAAAGGA 641S  G  Q  I  W  R  H  M  F  H  C  T  G  T  E  Q  H  M  G  D 1921AGTGGTCAGATCTGGAGGCACATGTTTCACTGCACTGGGACTGAGCAGCACATGGGAGAT 661C  P  V  T  A  L  G  A  T  L  C  S  A  G  Q  V  A  S  V  I 1981TGCCCTGTAACTGCTCTGGGCGCGACGCTGTGTTCTGCTGGGCAAGTGGCCTCTGTAATC 681C  S  G  N  Q  S  Q  T  L  S  P  C  N  S  T  S  L  D  P  T 2041TGCTCAGGAAATCAGAGCCAGACGCTATCCCCATGCAATTCAACATCTCTGGACCCAACA 701R  S  T  T  S  E  E  S  A  V  A  C  I  A  S  G  Q  L  R  L 2101AGATCTACCACTTCGGAAGAAAGTGCTGTTGCTTGTATTGCGAGTGGGCAACTTCGCCTG 721V  N  G  G  G  R  C  A  G  R  I  E  V  Y  H  E  G  S  W  G 2161GTAAATGGAGGCGGTCGCTGTGCTGGGAGAATAGAGGTCTACCATGAGGGCTCCTGGGGC 741T  I  C  D  D  S  W  D  L  S  D  A  H  V  V  C  R  Q  L  G 2221ACCATCTGTGATGACAGCTGGGACCTGAGTGATGCCCATGTGGTGTGCAGACAGCTGGGC 761C  G  V  A  I  N  A  T  G  S  A  H  F  G  E  G  T  G  P  I 2281TGTGGAGTGGCCATTAATGCCACTGGCTCTGCTCATTTTGGGGAAGGAACAGGGCCCATC 781W  L  D  E  V  E  C  N  G  K  E  S  H  I  W  Q  C  R  S  H 2341TGGCTGGACGAGGTGAACTGTAATGGAAAGGAATCTCATATCTGGCAATGCCGCTCACAC 801G  W  G  Q  H  N  C  R  H  K  E  D  A  G  V  I  C  S  E  F 2401GGCTGGGGGCAACACAACTGCAGACATAAGGAGGATGCAGGAGTTATCTGCTCAGAGTTC 821M  S  L  R  L  I  D  E  T  S  R  D  I  C  A  G  R  L  E  V 2461ATGTCTCTCAGACTGATTGATGAAACCAGCAGAGACATCTGTGCAGGGCGTCTTGAAGTT 841F  Y  N  G  A  W  G  S  V  G  K  S  N  M  S  A  T  T  V  E 2521TTTTACAATGGAGCTTGGGGCAGCGTTGGCAAGAGTAATATGTCTGCAACCACTGTGGAG 861V  V  C  R  Q  L  G  C  A  D  K  G  S  I  N  P  A  S  S  D 2581GTGGTATGCAGGCAACTGGGTTGTGCAGACAAGGGGAGCATCAACCCTGCATCTTCAGAC 881K  P  M  S  R  H  M  W  V  D  N  V  Q  C  P  K  G  P  D  T 2641AAGCCCATGTCCAGGCACATGTGGGTGGACAATGTCCAGTGTCCAAAAGGACCTGACACC 901L  W  Q  C  P  S  S  P  W  K  Q  R  V  A  S  S  S  E  E  T 2701TTATGGCAGTGCCCATCTTCTCCATGGAAACAGAGAGTGGCCAGTTCTTCAGAGGAGACC 921W  I  T  C  A  N  K  I  R  L  Q  E  G  T  S  N  C  S  G  R 2761TGGATCACATGTGCCAACAAGATAAGACTTCAAGAAGGAACCTCTAATTGTTCTGGACGT 941V  E  L  W  H  G  G  S  W  G  T  V  C  D  D  S  W  D  L  E 2821GTGGAGCTCTGGCACGGAGGTTCCTGGGGGACAGTGTGCGATGACTCCTGGGACCTTGAA 961D  A  Q  V  V  C  R  Q  L  G  C  G  P  A  L  E  A  L  K  E 2881GATGCACAAGTGGTGTGTCGACAGCTGGGCTGTGGCCCAGCATTAGAAGCACTAAAAGAG 981A  A  F  G  Q  G  T  G  P  I  W  L  N  D  V  K  C  K  G  N 2941GCAGCATTTGGTCAGGGGACTGGGCCTATATGGCTCAATGACGTGAAGTGCAAAGGGAAT 1001E  S  S  L  W  D  C  P  A  R  P  W  G  H  S  D  C  G  H  K 3001GAGTCTTCCTTGTGGGATTGTCCTGCTAGACCCTGGGGGCACAGTGACTGTGGCCACAAG 1021E  D  A  A  V  R  C  S  E  I  A  M  A  Q  R  S  S  N  P  R 3061GAAGATGCTGCTGTGAGGTGCTCAGAAATTGCAATGGCCCAAAGATCATCAAATCCTAGA 1041G  H  S  S  L  V  A  L  G  I  F  G  V  I  L  L  A  F  L  I 3121GGTCACTCATCCCTTGTTGCATTGGGGATCTTTGGTGTCATTCTTCTGGCCTTTCTCATC 1061A  L  L  L  W  T  Q  R  R  R  Q  Q  Q  R  L  T  V  S  L  R 3181GCTCTCCTCTTGTGGACTCAAAGGCGAAGACAGCAACAGCGGCTTACAGTTTCCTTGAGA 1081G  E  N  S  V  H  Q  I  Q  Y  R  E  M  N  S  S  L  K  A  D 3241GGAGAGAATTCTGTCCACCAAATTCAATACCGGGAAATGAATTCTTCCCTGAAAGCAGAT 1101D  L  D  V  L  T  S  S  E  Y  P  N  E  S  D  D  F  N  D  A 3301GATCTGGACGTGCTGACTTCCTCAGAATATCCCAATGAGTCAGATGATTTTAATGATGCT 1121G  L  I  S  V  S  K  S  L  P  I  S  G 3361GGGCTAATTTCTGTGTCTAAATCTCTTCCTATTTCTGGA 1MSKLRMVPHG NSGSADFRRC FALLCPSAVA VVSILSTCLM TNSLGRADKE MRLTDGEDNC SEQ ID61 SGRVEVKVQE EWGTVCNNGW GMDEVSVICR QLGCPTAIKA AGWANSRAGS GRIWMDHVSCNO: 48 121RGNESALWDC KHDGWGKHNC SHQQDAGVTC SDGSSLEMRL MNGGNQCSGR IEVKFQGQWG 181TVCDDNFNID HASVVCKQLE CGSAVSFSGS ANFGEGSGPI WFDDLVCSGN ESALWNCKHE 241GWGKHNCDHA EDVGVICLDG ADLSLRLVDG VTECSGRLEV KFQGEWGTVC DDGWDSNDAA 301VVCKQLGCPT AVTAIGRVNA SEGSGHIWLD NLSCQGDESA LWQCRHHEWG KHYCNHNEDA 361GVTCSDGSDL ELRLVGGGSR CAGTVEVEIQ KLLGKVCDRG WGLKEADVVC KQLGCGSALK 421TSYQRYSKVK ATNTWLFLSR CSGNETSLWD CKNWQWGGLS CDHYEEAKVT CSAHREPRLV 481GGDIPCSGRV EVKHGDTWGT VCDSDFSLEA ASVLCRELQC GTVISILGGA HFGEGNGQIW 541AEEFQCEGQE SHLSLCSVAS RPDGTCSHSR DVGVVCSRYT EIRLVNGQSP CEGRVELKIL 601GNWGSLCNSH WDIEDAHVFC QQLKCGVALS IPGGAHFGKG SGQIWRHMFH CTGTEQHMGD 661CPVTALGATL CSAGQVASVI CSGNQSQTLS PCNSTSLDPT RSTTSEESAV ACIASGQLRL 721VNGGGRCAGR IEVYHEGSWG TICDDSWDLS DAHVVCRQLG CGVAINATGS AHFGEGTGPI 781WLDEVNCNGK ESHIWQCRSH GWGQHNCRHK EDAGVICSEF MSLRLIDETS RDICAGRLEV 841FYNGAWGSVG KSNMSATTVE VVCRQLGCAD KGSINPASSD KPMSRHMWVD NVQCPKGPDT 901LWQCPSSPWK QRVASSSEET WITCANKIRL QEGTSNCSGR VELWHGGSWG TVCDDSWDLE 961DAQVVCRQLG CGPALEALKE AAFGQGTGPI WLNDVKCKGN ESSLWDCPAR PWGHSDCGHK 1021EDAAVRCSEI AMAQRSSNPR GHSSLVALGI FGVILLAFLI ALLLWTQRRR QQQRLTVSLR 1081GENSVHQIQY REMNSSLKAD DLDVLTSSEY PNESDDFNDA GLISVSKSLP ISG

Example 11 Various Cell Lines are Rendered Permissive to North AmericanPRRSV Infection Following Transient Transfection with pCMV-susCD163v1

Porcine Kidney (PK032495), Norden Labs Swine Testicular (NLST-1), NordenLabs Dog Kidney (NLDK-1) were obtained from Pfizer Inc. and were grownat 37° C. and 5% CO₂ in growth media consisting of Dulbecco's ModifiedEagle Medium (DMEM, Invitrogen catalog number 11965) supplemented with5% fetal bovine serum (FBS), 1 mM sodium pyruvate, 2 mM L-glutamine andantibiotics. Cell lines Baby Hamster Kidney (BHK21), Norden Labs FelineKidney (NLFK-1), and Rabbit Lung (RL) were obtained from Pfizer Inc. andwere grown at C and 5% CO₂ in growth media consisting of Dulbecco'sModified Eagle Medium (DMEM, Invitrogen catalog number 11965)supplemented with 10% fetal bovine serum (FBS), 1 mM sodium pyruvate, 2mM L-glutamine and antibiotics. Vero cells were obtained from PfizerInc. and were grown at 37° C. and 5% CO₂ in growth media consisting ofMinimum Essential Medium Alpha (MEM, Pfizer Inc. formulation)supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine andGentamicin at 20 micrograms per mL. Cell culture wells (35 mm)containing approximately 1×10⁶ cells were transfected with 2 microgramsper well of plasmid pCMV-susCD163v1, in DMEM without FBS or antibiotics,using Lipofectamine 2000 (Invitrogen catalog number 11668-027) accordingto the manufacturer's instructions. Cell line RL was transfected with1.0 micrograms per well of plasmid pCMV-susCD163v1. A member of the PAMcell cDNA library without an insert, designated pPAMB (essentially andempty pSport plasmid vector), was used as a negative control plasmid. At24 hours post transfection wells were aspirated and washed twice withDMEM/5% FBS followed by infection with North American PRRSV isolateP129. Virus was allowed to adsorb in 0.5 ml growth media for a minimumof two hours, after which additional media was added to a final volumeof 2.0 ml and incubated overnight. The virus was then removed, wellswashed twice with growth media, and fresh growth media added (2.0 ml perwell). A time zero sample of culture fluid was immediately taken inorder to determine the background level of infectious virus from theinoculum. At a minimum of 48 hours post infection cultures were screenedfor permissivity by removing culture fluids in order to assay viablevirus, and permissive cells in the monolayer were detected byfluorescent antibody assay (FA). The FA was completed by fixing themonolayer with 80% acetone and stained with FITC-conjugated monoclonalantibody SDOW17 (Rural Technologies Inc), which is specific for thePRRSV nucleocapsid protein. Viable virus was titrated by inoculatingdilutions of culture fluids onto MARC-145 cells. Table 5 shows theresults of virus infection by FA and the presence of progeny virus foreach cell line tested.

Failure to detect progeny virus from some cell lines may be the resultof low virus titer in the cell culture fluids, below the assay's limitof detection. Permissivity of Vero cells to PRRSV infection wasaugmented by the expression of susCD163v1. Compared to the time zeromeasurement of background virus, there was nearly a two-log increase invirus titers in Vero cells transfected with pCMV-susCD163v1, whereasthere was less than a one-log in titer increase in cells transfectedwith negative control plasmid pPAMB. All cell lines except NLDK-1 werepositive by FA for permissivity to North American PRRSV isolate P129infection after transfection with pCMV-susCD163v1.

TABLE 5 Screening of various cell lines for permissivity to NA PRRSVisolate P129 following transient transfection with pCMV-susCD163v1 orpPAMB Fluorescent Progeny Antibody assay virus produced TransfectedpCMV- pCMV- cell line susCD163v1 pPAMB susCD163v1 pPAMB BHK21 +++ − +++− PK032495 + − + − NKFK-1 + − + − NLST-1 + − − − NLDK-1 − − NT NT RL + −− − Vero ++ + ++ + +++ = Highly positive ++ = Moderately positive + =Slightly positive − = Not detectible NT = Not tested

Example 12 BHK21 Cells are Rendered Permissive to European PRRSVInfection Following Transient Transfection with pCMV-susCD163v1

Cell line Baby Hamster Kidney (BHK21) was obtained from Pfizer Inc. andgrown at 37° C. and 5% CO₂ in growth media consisting of Dulbecco'sModified Eagle Medium (DMEM, Invitrogen catalog number 11965)supplemented with 10% fetal bovine serum (FBS), 1 mM sodium pyruvate, 2mM L-glutamine and antibiotics. Cell culture wells (35 mm) containingapproximately 1×10⁶ cells were transfected with 2 micrograms per well ofplasmid pCMV-susCD163v1, in DMEM without FBS or antibiotics, usingLipofectamine 2000 (Invitrogen catalog number 11668-027) according tothe manufacturer's instructions. At 24 hours post transfection wellswere aspirated and washed twice with DMEM/5% FBS followed by infectionwith European PRRSV isolate 96V 198. Virus was allowed to adsorb for aminimum of 2 hours. The virus was then removed, wells washed twice withgrowth media, and fresh growth media added (2.0 ml per well). A timezero sample of culture fluid was immediately taken in order to determinethe background level of infectious virus from the inoculum. At a minimumof 48 hours post infection cultures were screened for permissivity byremoving culture fluids in order to assay viable virus, and permissivecells in the monolayer were detected by fluorescent antibody assay (FA).The FA was completed by fixing the monolayer with 80% acetone andstained with FITC-conjugated monoclonal antibody SDOW17 (RuralTechnologies Inc), which is specific for the PRRSV nucleocapsid protein.Viable virus was titrated by inoculating dilutions of culture fluidsonto MARC-145 cells. As a result of the transient transfection of BKH21with pCMV-susCD163v1, cells were rendered permissive to European PRRSVisolate 96V 198 infection and yielded progeny virus.

Example 13 CD163 Genes from Multiple Animal Species Render BHK21 CellsPermissive to PRRS Virus Infection

BHK21 cells grown in DMEM (Invitrogen catalog number 11965) supplementedwith 10% fetal bovine serum, 1 mM sodium pyruvate, and antibiotics, wereused in transient transfection experiments. Before transfection cellswere washed once with OptiMEM (Invitrogen) without serum or otheradditives. Lipofectamine 2000 (Invitrogen) was used in all transfectionexperiments according to the protocol provided by the manufacturer. Thetransfection mixture consisted of 10 microliters of Lipofectamine 2000and 2-3 micrograms of DNA per 35 mm well. After overnight incubation,transfection medium was removed and cells were infected with PRRSVisolate P129. Infection was allowed to progress for 24-48 hours, whencells were fixed with 80% acetone and stained with monoclonal antibodySDOW17 conjugated with FITC (Rural Technology Inc., Brookings, S. Dak.).Staining of the nucleocapsid protein was visualized under a fluorescencemicroscope. Table 6. Transient transfection of BHK21 cells with variousCD163 genes renders them permissive to PRRS virus infection

TABLE 6 Plasmid backbone CD163 gene PRRSV infection (FA) pCMV-ScriptSwine CD163v1 +++ pRSV-Script Swine CD163v1 +++ pcDNA3.1D Swine CD163v2++ pcDNA3.1D Human CD163v2 ++ pcDNA3.1D Mouse CD163v3 + pcDNA3.1DAfrican green monkey +++ (MARC-145 cell) CD163v2 pcDNA3.1D Vero cellsCD163v7 +++ pcDNA3.1D DH82 cell CD163v2 +++ +++ = Highly positive ++ =Moderately positive + = Slightly positive

Example 14 Generation of PRRSV-Permissive BHK21 Stable Cell Lines UsingpCMV-susCD163v1

BHK-21 cells were grown in Dulbecco's Modified Eagle Media (DMEM)supplemented with 10% fetal bovine serum, 1 mM sodium pyruvate, andantibiotics. For transfection, cells were seeded at approximately 90%confluency in 6 well plates and incubated over night at 37° C. in 5%CO₂. Cells were transfected with pCMV-susCD163v1 DNA using Lipofectamine2000 (Invitrogen) according to the manufacturer's instructions. One dayafter transfection the cells were trypsinized and re-seeded in 96 wellplates in a dilution series. To select for stable transfectants, themedia was supplemented with 1 mg/ml Geneticin (G418 sulfate, Invitrogencatalog number 10131-027) from this point forward. Medium was changedevery 3-5 days. Plates were cultured until those wells with coloniesderived from single cells reached confluency, at which point the plateswere trypsinized and seeded into duplicate 96 well plates. One of theduplicate 96 well plates was infected with PRRSV isolate P129 and clonespermissive to infection were identified by staining with FITC conjugatedmonoclonal antibody SDOW17. Positive clones were then expanded from thesecond duplicate plate. To ensure homogeneity the positive cultures weresingle-cell cloned by limiting dilution. At each cloning the subclonesthat displayed robust growth and high PRRSV permissivity were chosen forexpansion. Three clones designated BHK/CMV/v1 #3, BHK/CMV/v1 #5, andBHK/CMV/v1 #12 (FIG. 6) were selected. These cell lines have maintainedthe permissive phenotype through 20 passages.

Example 15 Generation of PRRSV-Permissive BHK21 Stable Cell Lines UsingpRSV-susCD163v1

BHK-21 cells were cultured as described in Example 14. BHK-21 cells weretransfected with pRSVsusCD163v1 using Lipofectamine 2000 as described inExample 14. Cloning of transfected cells and screening for permissiveclones was performed essentially as described in Example 14. From theoriginal cloning 3 single cell clones were identified as permissive andwere subsequently recloned two more times to ensure homogeneity and toattempt to isolate subclones of higher permissivity (see FIG. 7). Theresulting cell lines were named BHK/RSV/v1, #2, #3, and #4. All of theseclones have maintained the permissive phenotype through the highestpassage tested (passage 11 for clone #2, passage 7 for clone #3, andpassage 5 for clone #4).

Example 16 Generation of PRRSV-Permissive Feline Kidney Stable CellLines Using pCMV-susCD163v1

Parental Norden Labs Feline Kidney (NLFK) cells were grown at 37 degreesC. and 5% CO₂ in Dulbecco's Modified Eagle Medium (Invitrogen catalognumber 11965-092) supplemented with 10% fetal bovine serum, 1 mM sodiumpyruvate, and antibiotics. Several 35 mm wells containing approximately2×10⁶ cells each were transfected with 4 micrograms per well ofpCMV-susCD163v1, in OptiMEM, using Lipofectamine 2000 (Invitrogencatalog number 11668-027) according to the manufacturer's instructions.After overnight incubation, cells were removed from the substrate usingAccutase (Innovative Cell Technologies, catalog number AT104) diluted inmedium, and seeded into three 96-well plates at three densities(approximately 2×10², 2×10³, and 2×10⁴ cells per well). The cells wereallowed to settle overnight at 37 degrees C. before beginning selectionof stable transformants. The next morning medium was replaced with 100microliters/well fresh medium containing 500 micrograms/ml Geneticin(G418 sulfate, Invitrogen catalog number 10131-027) to select for cellsexpressing the neomycin resistance gene. Medium was changed every 2 or 3days to maintain Geneticin potency. After 19 days of selection, the96-well plate with the lowest initial cell density (approximately 200cells/well) yielded 70 empty wells and 26 wells with one or morecolonies of G418-resistant cells (calculated number of resistantcells/well is 0.3, using the Poisson distribution). These 26 wells weresplit into duplicate wells and allowed to settle overnight. One set ofwells was infected with PRRSV isolate P129, incubated for 24 hours, thenfixed with 80% acetone and stained with FITC-conjugated monoclonalantibody SDOW17 (Rural Technologies Inc), which is specific for PRRSVnucleocapsid. Of the 26 clones, 8 contained some cells that wereinfected by PRRSV. One of these, designated “NLFK-CMV-susCD163v1-G4”,was clearly more permissive than the others with nearly 100% of thecells staining positive for viral antigen.

By cell passage number 5, there was some evidence of phenotypicheterogeneity in the NLFK-CMV-susCD163v1-G4 cell line. Therefore, thecells were single-cell cloned by limiting dilution in G418-containingmedium, starting with frozen stocks of NLFK-CMV-susCD163v1-G4 passage 4.Twelve such clones (“A”-“L”) were expanded for study. Of these, clonesNLFK-CMV-susCD163v1-G4F and NLFK-CMV-susCD163v1-G4L were notable fortheir ability to form discrete plaques (localized areas of CPE) wheninfected with PRRSV isolate P129 (see FIG. 8).

Example 17 Generation of PRRSV-Permissive Feline Kidney Stable CellLines Using pRSV-susCD163v1

Norden Labs Feline Kidney (NLFK) cells were grown at 37° C. and 5% CO,in Minimal Essential Medium Alpha Medium (Invitrogen catalog number12571-071) supplemented with 10% fetal bovine serum and antibiotics.NLFK cells were seeded in 6 well plates at approximately 90% confluencyand allowed to attach overnight. The cells were then transfected withplasmid pRSV-susCD163v1 using Lipofectamine 2000 (Invitrogen) followingthe manufacturer's instructions. After 24 hours the cells were cloned asdescribed in Example 14. Screening for PRRSV permissive cell clones wasperformed as described in Example 14. Four clones were selected from thescreening and were single cell cloned by limiting dilution two moretimes. Four clones named FK/RSV/v1 #1, FK/RSV/v1 #2, FK/RSV/v1 #3, andFK/RSV/v1 #4 were selected. These cell lines have maintained the PRRSVpermissive phenotype through at least 8 passages (see FIG. 9).

Example 18 Generation of PRRSV-Permissive Porcine Kidney Stable CellLines Using pCMV-susCD163v1

Parental Porcine Kidney (PK032495) cells were obtained from Pfizer Inc.and were grown at 37 degrees C. and 5% CO₂ in growth media consisting ofDulbecco's Modified Eagle Medium (DMEM, Invitrogen catalog number 11965)supplemented with 5% fetal bovine serum (FBS), 1 mM sodium pyruvate, 2mM L-glutamine and antibiotics. Tissue culture wells (35 mm) containingapproximately 1×10⁶ cells each were transfected with 2 micrograms perwell of plasmid pCMV-susCD163v1, in DMEM without FBS or antibiotics,using Lipofectamine 2000 (Invitrogen catalog number 11668-027) accordingto the manufacturer's instructions. After overnight incubation, cellswere washed with PBS and removed from the substrate using Accutase(Innovative Cells Technologies, catalog number AT104) and diluted ingrowth medium containing Geneticin (G418 sulfate, Invitrogen catalognumber 10131-027) at 1.0 milligram per ml and seeded into 96-well platesat various densities to ensure recovery of single cell clones afterGeneticin selection. Throughout Geneticin selection, media was changedapproximately every 3 to 5 days. After selection, wells containingsingle cell clones were expanded into duplicate 96-well plates andallowed to incubate until 100% confluency was achieved. One set of wellswas screened for PRRSV-permissivity by infecting with PRRSV isolate P129for a minimum of 48 hours. Eleven clones were found to be permissive forPRRSV. One of these, designated “PK-CMV-susCD163v1-A10”, clearlyretained the permissive phenotype after numerous passages (see FIG. 10).

Example 19 Generation of PRRSV-Permissive BHK21 Stable Cell Lines UsingpCMVScript-susCD163v2

Parental Baby Hamster Kidney (BHK21) cells were obtained from PfizerInc. and were grown at 37 degrees C. and 5% CO₂ in growth mediaconsisting of Dulbecco's Modified Eagle Medium (DMEM, Invitrogen catalognumber 11965) supplemented with 10% fetal bovine serum (FBS), 1 mMsodium pyruvate, 2 mM L-glutamine and antibiotics. Tissue culture wells(35 mm) containing approximately 1×10⁶ cells each were transfected with2 micrograms per well of pCMVScript-susCD163v2, in DMEM without FBS orantibiotics, using Lipofectamine 2000 (Invitrogen catalog number11668-027) according to the manufacturer's instructions. After overnightincubation, cells were washed with PBS and removed from the substrateusing Accutase (Innovative Cells Technologies, catalog number AT104) anddiluted in growth medium containing Geneticin (G418 sulfate, Invitrogencatalog number 10131-027) at 1.0 milligram per ml and seeded into96-well plates at various densities to ensure recovery of single cellclones after Geneticin selection. Throughout Geneticin selection, mediawas changed approximately every 3 to 5 days. After selection, wellscontaining single cell clones were expanded into duplicate 96-wellplates and incubated until 100% confluency was achieved. One set ofwells was screened for permissivity by infecting with PRRSV isolate P129and incubating for a minimum of 48 hours. Three clones were found to bePRRSV-permissive, and one of these, designated“BHK-CMVScript-susCD163v2-A9”, was chosen for further study (see FIG.11).

Example 20 Generation of PRRSV-Permissive BHK-21 Stable Cell Lines UsingpRSV-susCD163v2

BHK-21 cells were cultured as described in Example 14. BHK-21 cells weretransfected with the ligated pRSV-susCD163v2 DNA construct described inExample 5 using Lipofectamine 2000 (Invitrogen) following manufacture'sinstructions. Subsequent cloning and selection of PRRSV permissive celllines was performed as described in Example 14. Of 336 single cellclones screened, 129 were positive. Several of these cell clones havebeen passed up to 7 times and they have maintained the PRRSV permissivephenotype (see FIG. 12). These cell lines have been named BHK/RSV/v2,followed by a numerical clone number.

Example 21 Generation of PRRSV-Permissive Porcine Kidney Stable CellLines Using pCMVSeript-susCD163v2

Parental Porcine Kidney (PK032495) cells were obtained from Pfizer Inc.and were grown at 37 degrees C. and 5% CO₂ in growth media consisting ofDulbecco's Modified Eagle Medium (DMEM, Invitrogen catalog number 11965)supplemented with 5% fetal bovine serum (FBS), 1 mM sodium pyruvate, 2mM L-glutamine and antibiotics. Tissue culture wells (35 mm) containingapproximately 1×10⁶ cells each were transfected with 2 micrograms perwell of pCMVScript-susCD163v2 in DMEM without FBS or antibiotics, usingLipofectamine 2000 (Invitrogen catalog number 11668-027) according tothe manufacturer's instructions. After overnight incubation, cells werewashed with PBS and removed from the substrate using Accutase(Innovative Cells Technologies, catalog number AT104) and diluted ingrowth medium containing Geneticin (G418 sulfate, Invitrogen catalognumber 10131-027) at 1.0 milligram per ml and seeded into 96-well platesat various densities to ensure recovery of single cell clones afterGeneticin selection. Throughout Geneticin selection, media was changedapproximately every 3 to 5 days. After selection, wells containingsingle cell clones were expanded into duplicate 96-well plates andincubated until 100% confluency was achieved. One set of wells wasscreened for permissivity by infecting with PRRSV isolate P129 andincubating for a minimum 48 hours. One clone designated“PK-CMVScript-susCD163v2-D1” showed the PRRSV-permissive phenotype.

Example 22 Generation of PRRSV-Permissive BHK21 Stable Cell Lines UsingpcDNA3.1D-humCD163v2

Parental Baby Hamster Kidney (BHK21) cells were obtained from PfizerInc. and were grown at 37 degrees C. and 5% CO₂ in growth mediaconsisting of Dulbecco's Modified Eagle Medium (DMEM, Invitrogen catalognumber 11965) supplemented with 10% fetal bovine serum (FBS), 1 mMsodium pyruvate, 2 mM L-glutamine and antibiotics. Tissue culture wells(35 mm) containing approximately 1×10⁶ cells each were transfected with2 micrograms per well of pcDNA3.1D-humCD163v2, in DMEM without FBS orantibiotics, using Lipofectamine 2000 (Invitrogen catalog number11668-027) according to the manufacturer's instructions. After overnightincubation, cells were washed with PBS and removed from the substrateusing Accutase (Innovative Cells Technologies, catalog number AT104) anddiluted in growth medium containing Geneticin (G418 sulfate, Invitrogencatalog number 10131-027) at 1.0 milligram per ml and seeded into96-well plates at various densities to ensure recovery of single cellclones after Geneticin selection. Throughout Geneticin selection, mediawas changed approximately every 3 to 5 days. After selection, wellscontaining single cell clones were expanded into duplicate 96-wellplates and incubated until 100% confluency was achieved. One set ofwells was screened for permissivity by infecting with PRRSV isolateP129, incubated for a minimum 48 hours. Seven candidate clones werefound to be PRRSV-permissive. There was some evidence of phenotypicheterogeneity in each of the seven candidate clones, likely because theywere not clonal. Therefore, the candidate clones were single-cell clonedby limiting dilution in G418 containing medium. One single cell clonewith clear PRRS-permissivity was obtained and designatedBHK-cDNA3.1D-humCD163v2-H9.

Example 23 Generation of PRRSV-Permissive Feline Kidney Stable CellLines Using pcDNA3.1D-humCD163v2

Parental Norden Labs Feline Kidney (NLFK) cells were grown at 37 degreesC. and 5% CO, in growth media consisting of Dulbecco's Modified EagleMedium (DMEM, Invitrogen catalog number 11965) supplemented with 10%fetal bovine serum (FBS), 1 mM sodium pyruvate, 2 mM L-glutamine andantibiotics. Tissue culture wells (35 mm) containing approximately 1×10⁶cells each were transfected with 2 micrograms per well ofpcDNA3.1D-humCD163v2 in DMEM without FBS or antibiotics, usingLipofectamine 2000 (Invitrogen catalog number 11668-027) according tothe manufacturer's instructions. After overnight incubation, cells werewashed with PBS, removed from the substrate using Accutase (InnovativeCells Technologies, catalog number AT104), diluted in growth mediumcontaining Geneticin (G418 sulfate, Invitrogen catalog number 10131-027)at 500 micrograms per ml, and seeded into 96-well plates at variousdensities to ensure recovery of single cell clones after Geneticinselection. Throughout Geneticin selection, media was changedapproximately every 3 to 5 days. After selection, wells containingsingle cell clones were expanded into duplicate 96-well plates andincubated until 100% confluency was achieved. One set of wells wasscreened for PRRSV-permissivity by infecting with PRRSV isolate P129 fora minimum 48 hours. Five clones were found to be permissive. One ofthese, designated “FK-cDNA3.1D-humCD163v2-A6”, clearly displayed thepermissive phenotype (see FIG. 13). NLFK parent cells and one subcloneof FK-cDNA3.1D-humCD163v2-A6 were examined for the CD163 expression.Cells were fixed in 80% acetone and reacted with Goat anti-human CD163(R&D System at 1:200) for one hour following by washing with PBS. Forvisualization, donkey anti-Goat IgG conjugated with FITC (Biodesign Incat 1:100) were used. No specific fluorescence was detected in the NLFKparent cells as shown in FIG. 21A. The majority of the FK.A6.A2 subcloneshowed good fluorescent staining indicating the presence of CD163 (FIG.21B).

Example 24 Generation of PRRSV-Permissive Porcine Kidney Stable CellLines Using pcDNA3.1D-humCD163v2

Parental Porcine Kidney (PK032495) cells were obtained from Pfizer Inc.and were grown at 37 degrees C. and 5% CO₂ in growth media consisting ofDulbecco's Modified Eagle Medium (DMEM, Invitrogen catalog number 11965)supplemented with 5% fetal bovine serum (FBS), 1 mM sodium pyruvate, 2mM L-glutamine and antibiotics. Tissue culture wells (35 mm) containingapproximately 1×10⁶ cells each were transfected with 2 micrograms perwell of pcDNA3.1D-humCD163v2, in DMEM without FBS or antibiotics, usingLipofectamine 2000 (Invitrogen catalog number 11668-027) according tothe manufacturer's instructions. After overnight incubation, cells werewashed with PBS, removed from the substrate using Accutase (InnovativeCells Technologies, catalog number AT104), diluted in growth mediumcontaining Geneticin (G418 sulfate, Invitrogen catalog number 10131-027)at 1.0 milligram per ml, and seeded into 96-well plates at variousdensities to ensure recovery of single cell clones after Geneticinselection. Throughout Geneticin selection, media was changedapproximately every 3 to 5 days. After selection, wells containingsingle cell clones were expanded into duplicate 96-well plates andincubated until 100% confluency was achieved. One set of wells wasscreened for PRRSV-permissivity by infecting with PRRSV isolate P129 fora minimum 48 hours. Two clones were found to be permissive. One ofthese, designated “PK-cDNA3.1D-humCD163v2-B11” clearly showed thePRRSV-permissive phenotype.

Example 25 Generation of PRRSV-Permissive Feline Kidney Stable Cell LineUsing Ligated pRSV-Script MARC CD163v2

A non-cloning based procedure to generate microgram quantities of linearDNA suitable for use in generating stable cell lines expressing CD163from an RSV promoter was developed (FIG. 4). A similar process wasadapted to place simian CD163v2 from MARC-145 cells behind the RSVpromoter. The procedure involves the isolation and ligation of twopieces of DNA, one containing the neomycin gene and RSV promotercassette derived from pRSV-script, and the other containing the MARCCD163v2 coding sequence from pcDNA3.1D MARC CD163v2. Vector plasmidpRSV-Script was linearized with Hind III and Kpn I. Plasmid was firstdigested with Kpn I and was bunted with the Klenow fragment of E. coliDNA polymerase. This plasmid was then digested with Hind III immediatelydownstream of the RSV promoter. The pcDNA3.1D MARC CD163v2 clone wasdigested in the vector sequence downstream of the CD163 insert withEcoRV, and Hind III upstream of CD163. The CD163 coding sequence wasliberated from the vector. For each plasmid digestion the appropriatefragments were purified from agarose gels. A large-scale ligationreaction was performed as follows. Approximately 20 μg of each DNAfragment was incubated in a volume of 600 μL with 15 units of T4 DNAligase. The reaction was incubated at room temperature for 1 hour.Following ligation, a linear piece of DNA containing all of theappropriate elements was purified by agarose gel electrophoresis.Restriction enzyme digestion analysis was performed to confirm theauthenticity of each ligated fragment. Ligation of the two DNA fragmentsvia the cohesive Hind III termini resulted in the placement of the 5′sequences of the MARC CD163 gene downstream of the RSV promoter,allowing for directed expression of CD163 in mammalian cells. Onceisolated, the purified DNA was used to transfect selected mammalian celllines.

Norden Labs Feline Kidney (NLFK) cells were grown at 37° C. and 5% CO₂in DMEM supplemented with 5% fetal bovine serum and antibiotics. NLFKcells were seeded in 6 well plates at approximately 90% confluency andallowed to attach overnight. The cells were then transfected withligated plasmid pRSV-MARC CD163v2 using Lipofectamine 2000 following themanufacturer's instructions. After 24 hours the cells were cloned asdescribed in Example 12. Screening for PRRSV permissive cell clones wasperformed as described in Example 12. One clone was positive for PRRSVinfection and designated NLFK-MARC CD163 D4. This D4 clone hasmaintained the PRRSV permissive phenotype through 9 passages.

Example 26 Growth Kinetics of PRRSV Isolate NVSL 94-3 in RecombinantBHK-21 and NLFK Cells Stably Expressing susCD163v1 from the CMV Promoter

The amounts of progeny virus produced by PRRSV-infected BHK-21 or NLFKcells stably engineered to express susCD163v1 were quantitated. Fourcell lines expressing susCD163v1, BHK/CMV/susv1 #3, BHK/CMV/susv1 #5,BHK/CMV/susv1 #12, and FK/CMV/susv1 G4 were seeded at sub confluency in6 well plates and, after overnight incubation, were infected with theNVSL 94-3 isolate of PRRSV. MARC-145 cells were included in theexperiment for comparison. The cells were infected with virus at anm.o.i. of approximately 0.1. Virus was adsorbed for 60-90 minutes andwas removed. Cells were washed three times with PBS to remove residualvirus. One-milliliter aliquots were harvested from the cultures at12-hour intervals starting immediately after infection and continuedthrough 96 hrs. Fresh culture media was added to the cells at varioustime points to maintain a culture volume sufficient to prevent the cellmonolayer from drying out. Culture supernatants were stored at −80°until all samples were collected. The amount of PRRSV present in theculture supernatants was determined by plaque assay on MARC-145 cells.FIG. 14 shows that all CD163 expressing recombinant cell lines testedwere able to produce progeny PRRSV.

Example 27 Blocking PRRSV Infection with Anti-CD163 Antibody:Transiently Transfected Cells

BHK-21 cells, seeded in 24 well plates, were transiently transfectedwith the plasmid pcDNA3.1D-MARC-CD163v2 described in example 8, usingLipofectamine 2000 as described in example 14. After overnightincubation to allow expression of CD163, a titration of goat polyclonalantibody specific for human CD163 (R&D Systems, cat #AF1607) in PBS wasadded to the cells in a volume of 100 μl. As a control, equivalentamounts of normal goat IgG (R&D Systems, cat #AB-108-C) were used.Following a one-hour incubation at 37° C., the monolayers were infectedwith approximately 1×10⁷ pfu of a recombinant P129 strain of PRRSV thatexpresses GFP. The cell monolayers, with anti-CD163 antibody and PRRSV,were incubated at 37° C. for one hour at which time the virusinoculum/antibody mixture was aspirated, the cell monolayer washed oncewith PBS, and 1 ml of growth medium added to the wells. The cells wereincubated for 24 hours at 37° C. to allow PRRSV directed GFP expression.For analysis, the cells were trypsinized, resuspended in 500 μl of PBSand analyzed by flow cytometry to innumerate the PRRSV infected cellsvia GFP expression. For flow cytometry, uninfected BHK-21 cells wereused to set the baseline for fluorescence detection, and approximately100,000 cells were analyzed from each subsequent sample. The results ofthis analysis, shown in FIG. 15, show that the CD163 specific antibodywas able to significantly reduce the number of infected cells whencompared to cells incubated with normal goat IgG.

Example 28 Blocking PRRSV Infection by Anti CD163 Antibody: StablyTransfected Cells

The NLFK cells that stably express human CD163(FK-cDNA3.1D-humCD163v2-A6), described in Example 23, were seeded into24 wells plates. After allowing the cells to attach overnight, atitration of goat polyclonal antibody specific for human CD163 (R&DSystems, cat #AF1607) in PBS was added to the cells in a volume of 100μl. As a control, equivalent amounts of normal goat IgG (R&D Systems,cat #AB-108-C) were used. Following a one-hour incubation at 37° C., themonolayers were infected with approximately 1×10⁷ pfu of a recombinantP129 strain of PRRSV that expresses GFP. The cell monolayers, withanti-CD163 antibody and PRRSV, were incubated at 37° C. for one hour atwhich time the virus inoculum/antibody mixture was aspirated, the cellmonolayer washed once with PBS, and lmlof growth medium added to thewells. The cells were incubated for 24 hours at 37° C. to allow PRRSVdirected GFP expression. For analysis, the cells were trypsinized,resuspended in 500 μl of PBS, and analyzed by flow cytometry toinnumerate the PRRSV infected cells via GFP expression. Approximately100,000 cells were analyzed from each sample. The results of thisanalysis, shown in FIG. 16, show that the CD163 specific antibody wasable to significantly reduce the number of infected cells when comparedto cells incubated with normal goat IgG.

Example 29 Generation of PRRSV-Permissive Porcine Kidney Stable CellLines Using pRSV-susCD163v2

Porcine kidney cells (PK032495) were cultured as described in Example21. For transfection, cells were seeded in a 24 well plate at 80%confluency and allowed to recover overnight. Transfection of ligatedpRSV-susCD163v2 DNA described in Example 5 was performed usingLipofectamine 2000 (Invitrogen) following the manufacturer'sinstructions. Subsequent cloning and selection of PRRSV permissive cellswas performed essentially as described in Example 14. The initialcloning by limiting dilution failed to yield single cell derived clones,so 5 wells with PRRSV permissive cells were recloned by limitingdilution to yield clonal cell lines. 10 clones were selected for furtherstudy and one of these clones, PK-RSVScript-susCD163v2 #9 showed theability to support foci growth of PRRSV early after infection (see FIG.18).

Example 30 Generation of PRRSV-Permissive Feline Kidney Stable CellLines Using pRSV-susCD163v2

NLFK feline kidney cells were cultured as described in Example 17. Fortransfection, cells were seeded at approximately 80% of maximal densityin 24 well plates. After overnight incubation the monolayers weretransfected with ligation derived RSV/susCD163v2 (see example 5) usingLipofectamine following the manufacturer's instructions. Cloning of thetransfected cells and selection of PRRSV permissive cell clones wasperformed essentially as described in Example 14. Of the 67 cell clonestested for PRRSV permissivity, 20 were found to be positive. An exampleof the staining observed is shown in FIG. 19.

Example 31 Passage of PRRSV Isolate P201 in PK-RSVScript-susCD163v2Cells

Amplification of a PRRSV clinical isolate was performed as follows.Peripheral alveolar macrophage (PAM) cells were seeded at 5.4E6 cellsper 10 cm2 in a 6 well dish using OptiMEM media supplemented with 2%FBS. After 6 hours the media was aspirated and a 2 ml aliquot of serumharvested from a PRRSV infected pig was added to the cells. Following a90 minute adsorption, the serum inoculum was removed and replaced withOptiMEM. At approximately 40 post infection the supernatant washarvested and clarified with a 10 minute centrifugation. The supernatantwas directly used to infect PK-RSVScript-susCD163v2 clone #9 cells usinga 6 hour adsorption. After removal of the inoculum the cells were refedwith DMEM. The P201 virus was serially passaged on thePK-RSVScript-susCD163v2 #9 cell line using alternating infected cell andcell free supernatant passes. We observed that for efficient spread ofthe virus, the cells should be seeded at 50-70% confluency the daybefore infection, using flasks of cells that were kept atsub-confluency. To follow the progression of infection, each passage wasreplicated in multiple wells of identically infected cells and at eachday one of the wells was acetone fixed and stained with the FITC labeledmonoclonal antibody SDOW17. If the percentage of infected cells was notgreater than 50% and significant progression of foci development overthe prior days observations were not seen, the cells in an equivalentwell were trypsinized and passed to multiple fresh wells. These infectedcell passages were typically at a 1:4 split and sometimes included theaddition of an equivalent number of cells from an uninfected culture.Alternatively, if the SDOW17 staining revealed that the infected cellfoci had spread sufficiently to account for greater than 50% of thetotal cells, cell free supernatant was harvested and used to infectmultiple wells of freshly seeded cells (FIG. 20). After 11 passages theintervening cell passages were not necessary as the virus was able togrow to sufficient titer to allow consecutive cell free supernatantpassaging of the virus.

Example 32 Screening Various CD163 Cell Lines for Permissivity toVarious European and North American PRRSV Isolates

Various CD163 transgenic cell lines were assessed for permissivity tolow passage European and North American PRRSV isolates (see Table 7).Transgenic CD163 cell lines as described in earlier examples includedNLFK-MARC CD163 D4, PK-RSVScript-susCD163v2 clone #9 andPK-CMV-susCD163v1-A10. Each CD163 cell line along with cell linesMARC-145, parental feline kidney, parental porcine kidney cell lines(serving as controls) were planted onto 96-well tissue culture plates.Growth media was removed from monolayers and inoculated with 0.1 mL perwell of each PRRSV isolate. At day 3 post infection, the plates werefixed with 80% acetone and stained with FITC-conjugated monoclonalantibody SDOW17 (Rural Technologies Inc.) which is specific for thenucleocapsid. Results of the fluorescent antibody (FA) assay are inTable 7.

TABLE 7 FA results of screening of various CD163 cell lines forpermissivity to European and North American PRRSV isolates PRRSVIsolate^(a) CD163 Cell Line EU98V226 P129 P201 1151 94-3 IND5 NLFK-MARC++ + +++ +++ ++ ++++ CD163 D4 PK-RSVScript- + + ++ + + ++susCD163v2clone #9 PK-CMV- + + ++ ++ ++ ++ susCD163v1-A10 MARC-145 ++ ++++ + ++++ +++ Porcine Kidney − − − − − − (parental) Feline Kidney − − −− − − (parental) ^(a)All PRRSV isolates are North American exceptEU98V226 is a European isolate.

Example 33 Phorbol 12-Myristate 13-Acetate (PMA) Induction of CD163Renders Human U937 Cells Permissive to PRRSV Infection

Human U937 cells obtained from ATCC(CRL-1593.2) were propagated in RPMImedium containing serum and additives according to ATCC specifications.These cells are known to express CD163 when activated by PMA treatment(Gronlund et al., 2000). U937 cells were seeded in duplicate in wells ofa 6-well plate. One set of wells was treated with 100 ng/ml of PMA andthe other set was left untreated. Three days after PMA stimulation, onewell from each set was infected with the P129 isolate of PRRSV. Theother well from each set was fixed and stained for expression of CD163in an indirect immunofluorescent antibody assay using goat-anti humanCD163 (R&D System) and donkey anti-goat IgG conjugated with FITC(BioDesign International).

Untreated U937 cells continued propagation to high density 3 days afterinitial planting. PMA-treated U937 cells stopped propagating, becameenlarged, and attached to the surface of the culture wells. A smallfraction of untreated U937 were positive for CD163 staining, whereasalmost all PMA-treated U937 were positive for CD163 staining. Inuntreated U937 no PRRSV infected cells were observed. However, hundredsof PMA treated U937 cells became infected by PRRSV. This demonstratesthat non-permissive cells can be rendered permissive for PRRSV infectionfollowing chemical induction of CD163 expression.

Additional features and variations of the invention will be apparent tothose skilled in the art from the entirety of this application,including the detailed description, and all such features are intendedas aspects of the invention. Likewise, features of the inventiondescribed herein can be re-combined into additional embodiments thatalso are intended as aspects of the invention, irrespective of whetherthe combination of features is specifically mentioned above as an aspector embodiment of the invention. Also, only such limitations that aredescribed herein as critical to the invention should be viewed as such;variations of the invention lacking limitations that have not beendescribed herein as critical are intended as aspects of the invention.

It will be clear that the invention may be practiced otherwise than asparticularly described in the foregoing description and examples.

Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, are within thescope of the invention.

The entire disclosure of all publications cited herein are herebyincorporated by reference to the extent they are not inconsistent to thedisclosure herein.

1. A method of facilitating production of Porcine Reproductive andRespiratory Syndrome Virus (PRRSV) from a culture of vertebrate cells,comprising the steps of: (a) providing a recombinant vertebrate celltransfected with an exogenous CD163 polynucleotide that encodes apolypeptide having CD163 activity, so that expression of the polypeptidein said cell is increased; (b) contacting a culture of said cell withPRRSV virus under conditions which permit infection of the cells andgrowth of the virus; and (c) recovering virus from said culture.
 2. Themethod of claim 1, wherein the cell was previously PRRSV permissive andis rendered more PRRSV permissive.
 3. The method of claim 1, wherein thecell is selected from the group consisting of baby hamster kidney cells(BHK21), porcine kidney cells, feline kidney cells, avian cells, andswine testicular cells.
 4. The method of claim 1, wherein the PRRSV isof the European genotype.
 5. The method of claim 1, wherein the PRRSV isof the North American genotype.
 6. The method of claim 1, wherein thepolynucleotide encodes a polypeptide having a transmembrane domain, andat least 70% sequence identity to SEQ ID NO:2.
 7. The method of claim 1,further comprising the step of producing a vaccine from the recoveredvirus.
 8. The method of claim 7, wherein the vaccine comprisesinactivated virus.
 9. The method of claim 7, wherein the vaccinecomprises live attenuated virus.
 10. The method of claim 1, wherein saidexogenous polynucleotide encodes a fusion protein.